NOTES Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 126.96.36.199 on 10/25/17 For personal use only. NODULATION MUTANTS OF WHITE BEAN (Phqseolus vulgaris L.) INDUCED BY ETHYL-METHANE SULPHONATE 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 is 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 reportsinitialfindingsofnodulationmutants 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. 199 200 CANADIAN JOURNAL OF PLANT SCIENCE Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 188.8.131.52 on 10/25/17 For personal use only. 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 as described for M2 populations. RESULTS AND DISCUSSION 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 OAC Number Treated seed M, plants matured M, plants examined Mutant rype Super nodulation (nrsn) No. % Copious nodulation No. % Non-nodulation No. % Albino No. % Rico 4500 r'15 1655 Swan Valley 3000 467 3586 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 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 www.nrcresearchpress.com by 184.108.40.206 on 10/25/17 For personal use only. PARK AND BUTTERY - NODULATION MUTANTS OF BEAN 20r 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 ap- 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 ,?/sr4 plants 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 r.86%. 202 CANADIAN JOURNAL OF PLANT SCIENCE Progeny Tests Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 220.127.116.11 on 10/25/17 For personal use only. 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. 198s). 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: 503-508. 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 : 1 l5-l 17. Rennie, R. J. and Kemp, G. A. 1981. Selection for dinitrogen-fixing ability in Phaseolus vulgaris L. at two low temperature regimes. Euphytica 30: 87-95. Rennie, R. J. and Kemp, G. A. 1983. N2fixation in field beans quantified by rsN-isotope dilution. II. Effect of cultivars of beans. Agron. J.275:645-649. 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. S. J. PARK and B. R. BUTTERY Research Station, Agricuhure Canada, Harrow, Ontario, Canada NOR 1G0. Received 5 May 1987, accepted 2 July 1987.