Plant Breeding 105, 33—39 (1990)
© 1990 Paul Parey Scientific Publishers, Berlin and Hamburg
ISSN 0179-9541
Institut fiir Pflanzenbau und Pflanzenziichtung,
Georg-August-Univcrsitdt, Gottingen (Germany, F.R.)
Variation of Seed Glucosinolates in Lines of Brassica napus
K. K R A L I N G , G . ROBBELEN, W . THIES, M . HERRMANN and M. R. AHMADI
With 4 figures and 2 tables
Received March 15, 1990 I Accepted March 22, 1990
Abstract
The glucosinolate (GSL) pattern of 93 resynthesized
(resyn) rapeseed lines was examined over three years,
and five stable genotypes with distinct GSL profiles
were identified. Typically the resyn B. napus profile
exhibited progoitrin as the main GSL, but contained
sinigrin. The different GSL patterns of the four
deviating lines are discussed with respect to the
proposed biosynthetic pathway. Within the resyn
and further extensive breeding materials, screening
for Io'w lndolyl GSL contents resulted in finding one
genotype with an extremely low 4-hydroxy-gluco-
brassicin and glucobrassicin content. Furthermore,
other lines were identified, differing over a wide
range of indolyl GSL contents. The values were
stable over two years.
Key words: Brassica napus — resynthesized
rapeseed — glucosinolate content — biosynthetic
pathway — indolyl glucosinolate — genetic stability
More than 90 different glucosinolates (CSEs)
are known up to now in the genus Brassica
(FENWICK et al. 1983). The main GSE groups
occurring in seeds of B. napus are alkenyl- and
indolyl CSLs. In the seeds, gluconapin
(GNA), progoitrin (PRO), and glucobras-
sicanapin (GBN) are the dominating alkenyl
GSLs, and 4-hydroxy-glucobrassicin (4OH)
and glucobrassicin (GBC) are the major in-
dolyl GSLs. Several investigations have shown
that the glucosinolate pattern in B. napus is
rather uniform (ANJOU et al. 1977, ADAMS et al.
1983). Therefore, efforts to use glucosinolate
profiles for cultivar identification were unsuc-
cessful (ADAMS et al. 1985).
a s . Copyright Clearance Cenlc," Code Statement: 01 79-954 1/90/050 1-0033$02.50/0
The biosynthesis of the glucosinolates is far
from being clear in many details. From several
studies it is supposed that alkenyl GSLs are
derived from methionin and indolyl GSEs
from tiyptophan (UNDERBILL 1980). The al-
kenyl GSLs PRO and gluconapoleiferin
(GNL) may be produced by hydroxylation of
GNA and GBN, respectively. In the synthesis
of indolyl GSLs, GBC seems to be the precur-
sor of the hydroxylated, methoxylated, and
sulfated analogues (MGDANELL et al. 1988). In
order to find also genetical evidence for these
pathways, genotypes would be extremely
helpful exhibiting different GSL patterns in
particular those with one individual GSL
dominating.
Following the identification of alleles re-
sponsible for a reduced alkenyl GSE content in
the Polish cultivar 'Bronowski' and their trans-
fer into breeding material, so called 00-varie-
ties, i.e. low in erucic acid content in the seed
oil and low in GSE content of their meal, were
released after a surprisingly short time. But in
these 00-varieties depending upon the low total
GSE content, now the share of the^ indolyl
GSEs may reach over 50% (own unpublished
results). Genotypes with reduced indolyl GSE
contents, therefore, received the particular at-
tention of the breeders. Since the GSE pattern
is a rather uniform trait within the convention-
al breedmg material, a broad spectrum of
rapeseed Unes has been analyzed, in particular
genotypes resulting from resynthesis based on
divergent B. oleracea and B. campestris par-
ents. This analysis revealed some GSE profiles
uncommon in B. napus. The results are de-
scribed in this paper.
34
Materials and Methods
Plant material: 93 rapeseed lines derived from ex-
perimental resynthesis by interspecific crosses of
B. oleracea X B. campestris (GLAND 1982) were dril-
led in the field in small observation plots (3.2 m'') in
September 1986. In each resyn line at least 5 plants
were selfpoUinated using paper bags just before
flowering. The bags were removed after flowering,
and completely mature seeds were analyzed by
HPLG for their GSL contents (see Table 1). Geno-
types with deviating GSL profiles were planted in
the following two years in the same manner as in
1986, but with larger plant numbers. For compari-
son, varietal lines were examined for their GSL
pattern.
In order to identify genotypes with a low indolyl
GSL content, all B. napus germplasm of the Plant
Breeding Institute of the University of Gottingen
was analyzed in the summer of 1988.
Analytical methods: About 200 mg seeds were
homogenized in a mill (modified IKA mill). GSL
extraction was carried out in polypropylene cen-
trifuge tubes at 75°G in a waterbath. For the first
extraction step the homogenized seeds were heated
for 10 min in 2 ml 70% aqueous methanol and
200 ^(1 6mmol glucotropaeolin (GTL)/1 water as in-
ternal standard (prepared according to THIES 1988).
After centrifugation (5 min, 2,500 X g) the pellet
was extracted a second time, using 2 ml 10% aque-
ous methanol. 500 ^l\ of the combined extracts were
pipetted on the top of a small ion-exchange column
containing 20 mg Sephadex DEAE-A25 in the for-
miate form. The column was washed with 2 X 1 ml
of sodium acetate buffer (20 mmol/1; pH 4.0). Desul-
fation was achieved by 75 fd 0.5% (w/v) of purified
sulfohydrolase (E.G. 3.1.6.1, type HI, Sigma
Ghemical Go.) and an incubation time of 16 h at
39 °G. Desulfated GSLs were eluted with 1.5 ml
water. After filtration (0.45 ^m) 70 ;(l of filtrate
were used for the HPLG analysis.
Table 1. Alkenyl glucosinolate contents (/(mol/g seed) of 93 resyn genotypes of B. napus in 1987
Abbreviation Trivial name Side chain
SIN Sinigrin 2-Propenyl-
GNA Gluconapin 3-Butenyl-
GBN Glucobrassicanapin 4-Pentenyl-
PRO Progoitrin 2-Hydroxy-3-butenyl-
GNL Gluconapoleiferin 2-Hydroxy-4-pentenyl-
ALY Glucoalyssin 5-Methylsulphinylpentenyl-
total alkenyl GSLs
KRALING, ROBBELEN, THIES, HERRMANN and AHMADI
Desulfoglucosinolates were analyzed by using a
Waters HPLG equipment, consisting of two pumps
model 510, an autosampler 710B, a controller 721
and an UV-detector model 441 at a wavelength
setting of 229 nm. Data were transmitted to a Waters
data module M730 and to a Gommodore G128 com-
puter by using a Waters M777 Data Transfer Mod-
ule. Separation was carried out by using a Macherey-
Nagel Nucleosil G-18 column (200 X 4 mm i.d.,
5 |»m particle size) and a linear solvent gradient frotn
1% to 19% acetonitrile in water over 18.5 min,
followed by a linear gradient to 1 % acetonitrile over
2 min and 1.5 min at isocratic condition. The flow-
rate was 1.8 ml/min at 35°C.
The identification of glucoberteroin (BER) was
accomplished with the help of seeds from Berteroa
incana (L.) DG, which contained 62.0 //mol of this
GSL (= 70% of all GSLs) per g seed. This seed
material was kindly supplied by Dr. W. Dersch,
Institute of Plant Physiology, Gottingen.
Prescreening for low indolyl glucosinolate con-
tents was accomplished by using a newly developed
photometric test (TlllES 1990). In this procedure, a
red coloured coupling product of the indolyl GSLs
with p-diazobenzenesulfonic acid (Fluka 33490) is
obtained in the presence of o-phosphoiic acid and
determined by usmg a multichannel photometer
(Flow Laboratories, Titertek Multiskan MG) at a
wavelength setting of 510 nm.
Results
Altogether 93 resyn lines were examined for
their GSE pattern. Variation of the total GSE
content and of the relative concentration of
individual alkenyl GSEs is shown in Table 1.
The sinigrin (SIN) content reached a mean of
3.5% and a highest value of 13.9%. PRO
dominated with an average content of 51.1%,
whereas its hypothetical precursor GNA exhi-
Gontents
Minimum Maximum Mean
S.0 7.6 1.9
3.6 46.3 14.7
0.0 6.6 2.3
2.4 36.7 28.1
0.0 2.2 0.7
0.0 6.7 1.3
10.5 127.0 83.1
 Variation of Seed Glucosinolates in Lines of Brassica napus 35
% GSL

80 -

60 -

40 -

20 -

K5 H 89 H 80 H 200 H 143
Lines
Fig. 1. Glucosinolate patterns of five resyn genotypes

bited an average value of 2 6 . 8 % . Eine H89 was
striking with a G N A content as high as
8 4 . 2 % . In general, mean shares of the pent-
enyl GSEs G B N , G N E , and glucoalyssin
( A E Y ) with 4 . 2 % , 1.3%, and 2 . 4 % , respec-
tively, were rather low in comparison to the
b u t e n y l GSEs. Unexpectedly low was the total
G S E content of line H 143 with 10.5/<mol/g
seed. T h e remaining lines exhibited high total
GSL contents confirming earlier results
( G L A N D 1982). O u t of these 93 resyn lines 20
g e n o t y p e s were selected with rather deviating
G S L profiles and tested the following year.
D u e t o nonreproducibihty of the parental GSE
values of 1987 in 1988, only 14 resyn lines were
carried over in 1989. The final examination of
these lines for their major individual GSLs
over t h r e e years revealed five stable genotypes
with distinct GSE profiles (Fig. 1). The resyn
hne H 80 exhibited a higher SIN content.
whereas in H 89 the hydroxylation step (for-
mation of P R O ) was obviously reduced. In
line H 200, the pentenyl GSEs, in particular
G B N and AEY, were increased. Eine K 5
represented the typical resyn B. napus profile
with P R O as the main GSE, but also contain-
ing sinigrin. Finally, the resyn line H 143 was
found to contain a Iow total GSE content, but
with a relative high content of indolyl GSEs
(58.2 % I). O t h e r hnes being unstable in respect
of their GSE profile showed strong variation of
individual alkenyl GSEs from year to year and
plant to plant.
For the identification of genotj'pes with low
indolyl GSE contents the absolute measure
",fnnol/g seed" was used and not the relative
concentration due to the varying amotint of the
total GSEs in this material. Table 2 represents
the variation of the two analyzed indolyl GSEs
in resyn and breeding material. Neither in the

Table 2. Indolyl glucosinolate contents (/imol/g seed) of 93 resyn genotypes (res) and a large number of
breeding lines (bre) of B. napus in 1987

Abbreviation Trivial name Side chain Gontents

Minimum Maximum Mean

GBC Glucobrassicin 3-lndolylmethyl- 0.0 1.3 0.3 res

0.0 0.9 0.3 bre
4OH 4-Hydroxygluco- 4-Hydroxv-3-indolylmethyl- 2.3 9.7 7.6 res
brassicin 2.6 6.1 4.3 bre
36
resyn nor in the breeding material sources for
alleles were found conditioning sufficiently
low 4OH concentration. The resyn genotypes
all together exhibited a high level of 4OH as
compared to the conventional material. No
differences in the mean GBC content were
detected. Therefore, in 1988 additional germ-
plasm was investigated particularly to detect
material with low indolyl GSL contents. Fol-
lowing the prescreening of ca. 850 plants by
the newly developed rapid test towards low as
well as high values, 80 samples were analyzed
by HPEC for confirmation. With this more
exact method one line was found exhibiting an
extremely low 4OH and GBC content. Fur-
thermore, other lines differing over a wide
range of indolyl GSE contents were identified.
To examine whether the selected genotypes
were stable in respect of their indolyl GSE
contents, the materiai was again tested under
field conditions in 1989. Figure 2 represents
the 4OH and GBC contents in 1988 and 1989
of four selected genotypes and one 00-cultivar
('Eirabon'). Eine B 24 contained only
0.5 //mol indolyl GSEs within 1 g seed ( =
0.4 % of the total GSE content), which was
stable over two years. Eine WDE also exhi-
bited a clear reduction of the indolyl GSE
content (= 2.3% of the total GSL content)
compared to the cultivar 'Lirabon', whereas
hnes N C Q and E 98 exhibited higher values
(7.1% and 12.5%, respectively, of the total
GSL content).
Ijmol GSL / g seed
B 24 WDE Lirabon
Lines
Fig. 2. Range of indolyl glucosinolate content in the genotypes examined
KRALING, ROBBELEN, THIES, HERRMANN and AHMADI
Discussion
The screening of 93 resyn genotypes over three
years finally revealed five genotypes exhibiting
unusual glucosinolate patterns (Fig. 1):
The resyn line K 5 representing a typical
high glucosinolate "resyn Brassica napus pro-
file" differs from the traditional breeding mate-
rial (ROBBELEN and THIES 1980) only by a higher
SIN content.
In the resyn line H 89 the predominant
glucosinolate is GNA. Similar resyn forms
have been described by GLAND (1982). These
lines exhibited normal fertility, but had only
36 chromosomes. H 89 also was completely
fertile which was demonstrated by single plant
yields of more than 100 g in wide stands; but it
has been proven that this line possesses the
normal diploid chromosome set of 38 chromo-
somes. GLAND et al. (1981) as well as SANG and
SALISBURY (1988) identified such GNA profiles
in B. campestris, too. Thus, in H 89 the
B. campestris genes responsible for GSE-
biosynthesis are obviously dominating. Ac-
cordingly, our selected line is not only charac-
terized by a low level of PRO, but also by the
lack of GNE, the hydroxylated analogue of
GBN.
In the resyn line H 80, GNA and PRO were
found as the major GSE components. Here,
the hydroxylation rates for butenyl- and pent-
enyl GSEs are reduced as compared to the GSE
pattern typical for B. napus genotypes; the
Snm of all GSLs
GSL (year)
CZ] 4OH (88)
^ 4OH (89)
CZLH GBC (88)
B i GBC (89)
NCQ E 98
 Variation of Seed Glucosinolates in Eines of Brassica napus 37

same step, on the other hand, is enhanced in
H 89. Further on, the SIN content in H 80 is
increased indicating that in this resyn line
genes of both parental genomes are expressed.
Up to now, such a profile has never been
described in the literature. Considering the
hydroxylation rates in K 5, H 89, and H 80, it
is striking that in the case of a reduced butenyl-
hydroxylation rate also the pentenyl-hydroxy-
lation rate is correspondingly decreased. This
might indicate that only one enzyme is neces-
sary for both hydroxylation reactions.
In the resyn line H 200 concentrations of
GSLs with five C-atoms in the side chain are
equally increased. Not only the biosynthesis of
the thio-GSLs GBN and GNL, but also the
synthesis of the sulfinyl GSL ALY is en-
hanced. After chain elongation and the ex-
change of the amino acid function, the first
pentenyl GSL to be expected in the biosynthe-
tic pathway (cf. Fig. 3) should be glucoberte-
roin (BER). In accordance with the hy-
pothesis, the amount of BER is also consider-
ably increased in the seeds of H 200: 1.9 /imol/
g seed were detected instead of 0.2 /.imol/g
which are usually found in B. napus seeds.
However, the HPEC chromatogramme,
shown in Fig. 4, demonstrates that BER can-
not be completely separated from gluconastur-
tiin (NAS). SANG and SALISBURY (1988), did not
describe such diversity of pentenyl GSLs in
their cultivars, the type 1 of which contained
GBN only.
The resyn line H 143 had the lowest total
GSL content together with a remarkably high
relative and absolute indolyl GSL content.
Profiles described for low-glucosinolate lines
by ADAMS et al. (1985) and SANG and SALISBURY
(1988) differed from H 143 by exhibiting PRO
as the dominant GSL, whereas H 143 con-
tained more than 50% 4OH. Therefore, this
line should be helpful in studies analyzing of
4OH as a repellent.
Our investigation demonstrates the exist-
ence of different alleles involved in the biosyn-
thesis of GSLs in B. napus, although differ-
ences in the GSL pattern between cultivars
were not found (ADAMS et al. 1983, 1985).
However, ADAMS et al. (1989) investigating
swede were able to distinguish nine cultivars
by comparing the relative proportions of indi-
vidual (JSLS in medulla extracts.
Due to the low alkenyl GSL content in 00-
cultivars the percentual share of indolyl GSLs
increases and, therefore, variation of indolyl
GSL content becomes more important. Our

^ 1) 1)
^^ 3L-Metiilorimc ;^- 4U-lv

Hvdrnxv- ^' Progoitrin Gluconapoleiferin

Alkeml-GStj: 2)

14)
11 4)
1 1

Alkenvl-GSt.i Sinigrin Gluconapin Glucobrassicanapin

(-S-)
f'
— ^ Glucoibervirin
1
—>-
t"
Glucoenicin ' >• Glticobeneroin
1 >
1 1
1 4)

Y T Y
SuKnvt-GSt..! Glucoiberin Glucoraphanin Glucoalyssin
(SO-) 1 1 1
1
|4) |4)
Y' Y Y
(-SO2-) Glucocheirolin Glucoerysolin N.N.

PrommUGSU Pentenvl-GSt s
Fig. 3 . Hypothetic pathway for the biogenesis of alkenyl-glucosinolates in seeds of Brassica species. The steps
involved are: 1) chain elongation, 2) exchange of the "amino acid function" by a "glucosinolate function",
3) loss of the methyl group and introduction of a double bond, 4) oxidation
38
Fig. 4. Chromatogramme obtained after HPEC sep-
aration of seed glucosinolates of the resyn line H 200
screening for indolyl GSEs revealed a wide
range of 4OH contents from about 0.5 to
10 ^mol per g seed (Fig. 2). One lme (B 24)
developed from a cross between a yellow
seeded resyn genotype and a brown seeded
B. napus line was found with an extremely low
indolyl GSE content. Meanwhile, further
studies have shown that there is no correlation
between the traits yellow seed coat and low
indolyl GSE content. Such a genotype is the
first of its kind which has been described. In
the present 00-cultivars, only aliphatic GSEs
are reduced by alleles derived from the cultivar
'Bronowski'. Therefore, the usual OO-forms
are characterized by a rather high amount of
indolyl GSEs, since these GSEs become in-
creasingly important the more the alkenyl
GSEs are eliminated. Our line B 24 will be
most helpful for further reduction of the total
GSE content in rapeseed cultivars. However,
genetic studies are necessary to develop ap-
propriate breeding strategies. Since the alkenyl
KRALING, ROBBELEN, THIES, HERRMANN and AHMADI
GSE content is controlled at least by three
genes (EEIN 1970) and determined by the ma-
ternal genotype rather than by the genotype of
the embryo (KONDRA and STEFANSSON 1970),
only one recessive low alkenyl type is detect-
able out of 100 F3 seeds harvested from Fi
plants. On the other hand, for the indolyl
GSEs BucHNER (1988) supposed that the hy-
droxylation of GBC to 4OH occurs in the seed
and is under the control of the embryo; but
GBC may be produced in the pods and is then
transported into the seeds. Therefore, in our
low indolyl GSE type the transport mechanism
or the hydroxylation step might be blocked
implying different selection strategies. At any
rate reduction of the total GSE content by
convential backcrossing seems to be the most
appropriate method at present. Due to the high
alkenyl GSE content of B 24, a greater popula-
tion size is necessary to screen for both GSE
types. For breeding cultivars with low total
GSE contents, the segregating population
should be prescreened by using the photo-
metric test detecting indolyl GSEs. By this
means, selected genotypes can be backcrossed
with 00-genotypes without considering the al-
kenyl GSL content.
Zusammenfassung
Unterschiede in den Samenglucosinolaten
von Rapslinien (B. napus)
Im Herbst 1987 wurden die Glucosinolatmu-
ster von 93 resynthetisierten (resyn) Linien be-
stimmt. Die resyn Einie K 5 enthalt Sinigrin
und reprasentiert damit das typische resyn
B. napus-Muster mit Progoitrin als Haupt-
glucosinolat. Nach dreijahriger Prtifung wur-
den vier Einien mit abweichenden GSE-Mu-
stern identifiziert. So zeichnen sich H 89
durch einen hohen Gluconapingehalt und
H 143 durch einen niedrigen Gesamt-GSE-
Gehalt aus. Die resyn Einie H 80 weist einen
hohen Sinigringehalt auf, und in H 200 waren
die Pentenyl-GSEe erhoht. Die verschiedenen
GSE-Muster werden in bezug auf den bisher
noch hypothetisehen Biosyntheseweg dis-
kutiert.
Im ersten Ansatz blieb eine umfangreiche
Auslese auf niedrige Indolyl-GSE-Gehalte er-
folglos. In weiteren Untersuehungen wurde
jedoch ein Genotyp gefunden, der uber zwei
Jahre extrem niedrige 4-Hydroxy-Glucobras-
Variation of Seed Glucosinolates in Lines of Brassica napus
sicin- und Glucobrassicin-Gehalte aufwies.
Zusatzlich konnten weitere Einien mit ver-
schiedenen Indolyl-GSE-Gehalten nach-
gewiesen werden.
Support of this work by the Bundesministerium fiir
Ernahrung, Landwirtschaft und Forsten (BMFL),
Bonn, and the Gemeinschaft zur Forderung der
privaten deutschen landwirtschaftlichen Pflanzen-
zliehtung (GFP), Bonn, is gratefully acknowledged.
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Authors' addresses: Dr. K. KRALING, Prof. Dr. G.
ROBBELEN, Prof. Dr. W. THIES, Dipl.-Biol. M.
HERRMANN, Institut fiir pflanzenbau und Pflanzen-
ziichtung, Universitat, Von-Siebold-Str. 8, D-3400
Gottingen (Germany, F.R.); Dr. M. R. AHMADI,
Seed and Plant Improvement Institute (S.P.I.I.),
Mard-Abad Avenue, Karadj (Iran).