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Can. J. Plant Sci. Downloaded from by on 10/25/17
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Seed'of the OAC Rico and Swan Valley cultivars of bean (Phaseolus vulgaris L.) was
treated with 0.04 M ethyl-methane sulphonate (EMS). Screening of M, populations
in the presence of 8 mM nitrate + 2 mM ammonium, which substantially inhibited
nodulation of the parental cultivars revealed nitrate tolerant supernodulating (ntsn),
copious nodulating and non-nodulating mutants. One ntsn mutant from 175 Mt lines
of OAC Rico and two ntsn mutants from 467 M, Iines of Swan Valley were obtained.
M, progenies derived from the Rico and the one fertile Swan Valley nrsn-mutants (Mr)
bred true.
Key words.: Nodulation, dry edible (navy, common) bean, EMS, Phaseolus vulgaris
[Formation de mutants de nodulation chez Ie haricot blanc (Phaseolus vulgaris L.)
par un traitement au m6thane sulfonate d'6thyle.l
Titre abr6g6: Mutants de nodulation de haricot.
La semence des cultivars OAC Rico et Swan Valley du haricot blanc (Phaseolus vulgaris L.) a 6te ftaill-, au m6thane sulfonate d'6thyle (MSE) 0,04 M. Des essais s6lectifs
de populations M, en pr6sence de 8 mM de nitrate * 2 mM d'ammonium, qui inhibe
consid6rablement la nodulation des cultivars parentaux, a rdv6l6 la pr6sence de mutants
supernodulants (nrsn), fortement nodulants et non nodulants tol6rants au nitrate. Un
M, de OAC Rico et 2 mutants r?trn issus de 467 lign6es
M, de Swan Valley ont ainsi 6t6 obtenus. Les descendances M, issus du parent Rico
et le seul mntant ntsn Swan Valley fertile (Mr) sont stables dans leur descendance.
mutant,?tsr? issu de 175 lign6es
Mots cl6s: Nodulation, haricot sec de consommation (petit haricot blanc, commun),
MSE, Phaseolus vulgaris
in N2-fixation
The common bean (Phaseolus vulgaris L.)
with ethyl-methane-sulfonate (EMS) solution
generally considered poor
is a simple way of inducing high frequencies
of mutation of various types. This paper
(Vincent 1974) and shows variable response
to inoculation (Graham and Rosas 1977;
Ruschel et al. 1982). Genetic variability for
nodulation and N2 fixation of held bean has
been demonstrated, but these variants vyith
enhanced nodulation and N2 fixation were
of white bean mutagenized by EMS.
About 4500 and 3000 seeds of two white
bean cultivars, OAC Rico and Swan Valley,
were soaked in tap water for 12 h at room
temperature on 5 Sept. 1985. These seeds
selected in the absence of or at low rates
of nitrate (Rennie and Kemp 1981, 1983). were transferred to 0.b4 M aqueous solution
Nitrate added at the time of sowing suppressed (adjusted to pH 7 with phosphate buffer) of
N2 fixation; some host-symbiont relation- gMS (ethyl-methane-sulphonate) and
ships were more sensitive to N-fertilization incubated for 6 h at22"C. TLe mutagenized
than others (Westerman et al. l98l). Since M1 seeds were rinsed in running tap water
mutants that efficiently nodulate in the for 30 min and were sown in soil beds (steampresence of high nitrate concentration have crcri1izerl ? .,tr eqrlier)
Leen obtained ror pea (Jacobsen re84)
and ;.}'jtTL:"Jff]i]',i?-:X lilt'ii11lil'i;
al' 1985), it should be pos- the two cultivars were also sown as controls.
sible to obtain white bean mutants which The number of seedings
emerged was
soybean (carrol et
nodulate more profusely and are less sensicounted' and compared with the control' M'
rve ro nlrrate rnnlDltron. Treatment of seed
seed from individual plants was harvested as
Can. J. Plant Sci. 68: 199-202, (Jan. 1988)
plants matured.
Can. J. Plant Sci. Downloaded from by on 10/25/17
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For screening of nodulation in M2 populations, 10 seeds. from each M2 line were
sown in black plastic pots (25 cm diameter
and25 cm deep) filled with a mixture of vermiculite and perlite at | :2 by volume. Seeds
were placed at 2 cm depth in holes marked
for planting, and inoculated with an effective
strain of Rhizobium phaseoli, TAL 182,
applied as a turbid yeast-mannitol broth
culture (ca. 107 cells ml-l) at seed level
before covering the seed with the pot medium.
Pots were watered daily with tap water until
seedlings emerged. The number of albino
seedlings was recorded. At the unifolioliate
stage nitrate treatment was imposed by
watering daily with a nutrient solution containing 4 mM Ca(NO3)2, 2 mM NH4CI,
1.2 mM KH2PO4, 1.0 mM MgSOa, 1.5 mM
K2SOa, 0.08 mM Fe citrate, plus a balanced
micro-nutrient supplement. Approximately
330 mL of the nutrient solution was given to
each pot once a day. Plants in control pots
received nutrient solution lacking combined
N. Plants were cultured in the presence of
nitrate for about 3 wk. These 4-wk-old plants
were carefully removed from the pot medium
and nodulation was rated visually as: 0 : no
nodulation, 1 : less than 5 nodules per plant,
2 : 5-10 nodules per plant, 3 : sparsely
nodulated (10-20 nodules per plant), 4 :
many nodules (20-50 nodules per plant), and
5 : profusely nodulated (when observed in
the presence of nitrate, referred to as the
"nitrate-tolerant super nodulation" (nrsn)
characteristic). Selected plants were repotted
and grown for seed.
For M3 progeny tests of ntsn M2 plants,
seedlings were grown and evaluated
described for M2 populations.
M1 Populations
Three weeks after sowing, 5.3 and 34% of
the mutagenized seed of the OAC Rico (Rico)
by comparing the 34% emergence of the
mutagenized SV population with that of
Mohan et al. (1980), who used 5O% emergence as a criterion, 0.04 M EMS was an
appropriate concentration.
Plant growth ranged from cessation of
growth at the unifolioliate stage to nearly
normal height growth. Most of the severely
affected plants died gradually before they
reached seed production. Four percent and
16% of the EMS-treated plants of Rico and
SV produced mature M1 plants (Table 1)
with variable amounts of M,
by each plant.
seed produced
Table 1. Number of M, plants that reached maturity,
and numbers and percentages of lines that exhibited nodu-
lation mutations in M, populations derived from EMStreated white bean cultivars OAC Rico and Swan Valley,
at Harrow. 1985*1986
Pooulations derived from
Treated seed
M, plants matured
M, plants examined
Mutant rype
Super nodulation (nrsn)
Copious nodulation
Swan Valley
1 (7)t 2 (4)I
0.6(0.4) 0.4(0.03)
rs (27) 26 (39)
8.6(1.7) s.6(1.1)
5 (6)
4 (8)
2.9(0.4) 0.9(0.2)
6 (10)
2 (4)
1. 1(0.3) l.3(0.3)
fValues in parentheses refer to numb€r or percent of M,
plants obtained from the selected M, lines.
M2 Generation
The two parental cultivars, OAC Rico
and Swan Valley (SV) populations had
emerged, compared to 4l and 94% emergence for the untreated seed ofthe same cul-
Swan Valley, produced many nodules (visual
scores of 3-4) in the absence of nitrate (control), but with added nitrate, nodulation was
considerably suppressed (visual score about
tivars, respectively. The Rico population was
severely affected by the treatment. However,
2). One of the 175 M1 lines derived from
OAC Rico (No. 32) and two of the 467 M1
Can. J. Plant Sci. Downloaded from by on 10/25/17
For personal use only.
Fig. 1. Nodulation of common
bean (P. vulgaris L.) cv. OAC Rico (right) and EMS-induced NTSN
(nitrate tolerant supernodulating)-mutant (left) selected from M, populations in the presence of 4 mM
Ca(NO)r * 2 mM NH4C1. Plants grown for 4 wk in vermiculite/perlite mixture in greenhouse.
lines derived from Swan Valley (Nos. 145 and
419) segregated in M, to produce ntsn
mutants that nodulated profusely (nodulation
score of 5) in the presence of combined N
(Table 1). The number ofnodules produced
by the ntsn mutants was much larger than that
of the parental cultivars grown with added
nitrate in the nutrient solution (Fig. 1). The
size of the nodules was similar in the ntsn
mutants and the wild type.
parative purposes in both Rico and SV populations (Table l). Also, the frequencies of ntsn
mutants in these two bean populations (0.58
and,O.43%) were very similar to those found
after EMS treatment of other legume crops:
0.457o in pea (Jacobsen 1984) andO.6% in
soybean (Carrol et al. 1985).
Two other types of nodulation variants were
observed: copius nodulating and non-nodulating types (Table 1). The copious nodulating
Rico line no. 32 (Rico32M1) segregated
type (nodulation score of 4) produced
of 32 plants tested in
preciably more nodules than the wild type
M2. SV lines no. 145 (SV145M1) and no.
419 (SV4I9M1) segregated I ntsn plant out
of 9 plants, and 3 ntsn plants out of l0 plants
(nodulation score of 2) in the presence ofhigh
concentration of nitrate. The non-nodulating
type bore no nodules or occasionally a few
nodules (nodulation scores of 0-l). Mean
mutation rate for the copious nodulating type
of the two populations was 7 % on the basis
seven /,/sn plants out
in M2. All three
of SV4I9M2
failed to produce M3 seed because they were
sterile, though plant growth was vigorous.
Frequency of ntsn mutants was approximately one-half to one-third of that of albinos
which were observed in the M2 for com-
of lines. Mean mutation rate for the nonnodulating type of the two populations was
Progeny Tests
Can. J. Plant Sci. Downloaded from by on 10/25/17
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M3 progenies (a total of 128) grown from the
seeds obtained from two Rico ntsn mutant
M2 lines, and from one SV ntsn mutantM2
line, produced nodules as profusely (nodulation score of 5) as their ntsn M2 parents
did. This indicated that the ntsn characteristic
bred true. Root and shoot growth of Rico ntsn
M3 plants was similar to that of OAC Rico.
However, overall growth of SV ntsn M3
plants was suppressed though the super-
nodulating characteristic was fully expressed.
Mutation(s) affecting plant growth may have
occurred, or prolific nodulation in SVl45 ntsn
plants may have been at the expense of root
growth as shown in soybean (Carrol et al.
The nodulation mutants obtained in this
study are of considerable academic interest.
Furthermore, the ntsn or the copius nodulating trait could be of agronomic value in
improving nodulation and N2-fixation in
bean under held conditions. Studies characterizing the mutants and clarifying genetic
control are being undertaken.
We thank R. Armstrong and
L. A. Davison for
technical assistance.
Carrol, B. J., McNeil, D. L. and Gresshoff,
P. M. 1985. Isolation and properties of soybean
lGlycine rutx (L.) Merr. I mutants that nodulate
in the presence ofhigh nitrate concentration. Proc.
Nat. Acad. Sci. U.S.A. 82: 4162-4166.
Graham, P. H. and Rosas, J. C. 1977. Growth
and development of indeterminate bush and
climbing cultivars of Phaseolus vulgaris L. inocu-
lated with Rhizobium. Agric. Sci. (Camb.) 88:
Jacobsen, E, 1984. A new pea mutant efficiently
nodulating in the presence of nitrate. Pages 597
inC.Yeeger andW. E. Newton, Eds. Advances
in nitrogen fixation research. Proc. 5th Int. Symp.
Nitrogen Fixation. Martinus Nijhoff/Dr. W. Junk
Publ., The Hague, The Netherlands.
Mohan, D. P., Benepal, P. S., Sheikh, A. Q.'
and Ranyoppa, M. 1980. Determin4tion of
optimal mutagenic dose of ethyl methane sulfonate,
diethyl sulfate and ethidium bromide for beans
(Phaseolus vulgaris L. ). Bean Improv. Coop. 23 :
Rennie, R. J. and Kemp, G. A. 1981. Selection
for dinitrogen-fixing ability in Phaseolus vulgaris
L. at two low temperature regimes. Euphytica 30:
Rennie, R. J. and Kemp, G. A. 1983. N2fixation in field beans quantified by rsN-isotope
dilution. II. Effect of cultivars of beans. Agron.
Ruschel, A. P., Vose, P. B., Matsui, E.,
Victoria, R. L. and Saito, S. M. T. 1982. Field
evaluation of Nr-frxation and N-utilization by
Phaseolus bean varieties determined by l5N isotope dilution. Plant Soil 65:397-4O7.
Vincent, J.M. 1974. Root nodule symbiosis with
Rhizobium.Pages26-341rz A. Quispel, Ed. The
biology of nitrogen fixation. North Holland Publ.
Co., Amsterdam, The Netherlands.
Westerman, D. T., Kleinkopf, G. E., Porter,
L. K. and Leggett, G. E. 1981. Nitrogen sources
for bean seed production. Agron. J. 73:. 6@-664.
Research Station, Agricuhure Canada,
Harrow, Ontario, Canada NOR 1G0.
Received 5 May 1987, accepted 2 July 1987.
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