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Agriculture and Agri-Food Canada Greenhouse and Processing Crops Research Centre, Harrow, ON, Canada N0R 1G0
* Corresponding author (parks{at}agr.gc.ca)
The common bean (Phaseolus vulgaris L.) genetic stocks R69 (Reg. no. GS-245, PI 641003) and R99 (Reg. no. GS-246, PI 641004) are ineffective and nonnodulation mutants of navy bean cultivar OAC Rico, respectively. They were selected in a dry bean breeding program conducted by Agriculture and Agri-Food Canada Greenhouse and Processing Crops Research Centre at Harrow, ON. The mutant lines were obtained during the development of white, dry edible beans with improved nodulation and N2 fixation in the presence of nitrate.
About 4500 water-soaked seeds of OAC Rico were immersed in a solution of 0.04 M ethyl methane sulphonate (EMS) and incubated for 6 h at 22°C. The mutagenized M1 seeds were rinsed in running tap water for 30 min before sowing in a greenhouse in 1985 (Park and Buttery, 1988). The plants were selfed and M2 seeds were harvested individually from 175 M1 plants as they matured. For initial screening of nodulation in the M2 population in 1987, 12 seeds harvested from 75 individual plants were sown in black plastic pots filled with a mixture of vermiculite and perlite (1:2 v/v). Seeds were inoculated with Rhizobium leguminosasarum bv. phaseoli (Buchanan) Jordan strain TAL 182, applied as turbid yeast–mannitol broth culture (ca. 107 cell mL–1, 1 mL seed–1) at seed level before covering the seed with potting medium. Pots were watered until seedlings emerged and thereafter received 330 mL of N-free nutrient solution daily. After 4 wk, when nodulation was expected to be near maximum, plants were carefully removed from the potting medium and visually rated for nodulation (Park and Buttery, 1988). Putative M2 plants lacking effective nodules were replanted and given 5 mM nitrate to increase plant growth and to boost seed production. In 1988 we screened 95 additional M2 lines and the M3 and M4 plants from the 1987 tests in the same manner, except that 1 mM nitrate was added to the nutrient solution to increase seed production because plants were otherwise too chlorotic and weak. Putative nonnodulating or ineffectively nodulating mutant plants were identified, some of which either did not produced seed or did not breed true.
Progeny tests with M3 plants of two M2 families confirmed two nodulation mutants, R69 and R99. R69 was sparsely nodulated with tiny pale white nodules which appeared to be nonfunctional (ineffective) in N2 fixation as evidenced by chlorotic shoot growth. R99 produced very few tiny nodules or no nodules. Both R69 and R99 are presumed to be incapable of fixing N because in the absence of nitrate they both form small yellow shoots that die after 7 to 8 wk without setting seed. In the same test, the wild-type OAC Rico produced large pinkish nodules with four- to five-fold higher nodulation scores than those of the mutants. The M4 progenies of both mutants bred true for the nodulation characteristics (Park and Buttery, 1992). Subsequently R69 and R99 mutants were backcrossed to OAC Rico to BC4 to recover vigor and other agronomic characteristics of the wild type during 1992 through 1994. After the final backcrossing, BC4 F3 plants were bulked to form basic seed of the mutants.
Inheritance of R69 ineffective nodulation (IN) mutant allele and R99 nonnodulation (NN) mutant allele was determined in crosses with two wild-type OAC Rico navy bean and ‘Midnight’ black bean (Park and Buttery, 1994). Both nodulation characteristics, IN and NN, were controlled by different single recessive genes. Complementation tests, including with the nitrate-tolerant super nodulation (NTSN) mutants of R32 for which a gene symbol nts has been assigned previously (Park and Buttery, 1989), revealed that all three nodulation types were under nonallelic control with distinct function. The gene symbols nie and nnd-2 were assigned for IN and NN types, respectively (Park and Buttery, 1994). Recessive epistasis occurred with nie being epistatic to nts, and nnd-2 being epistatic to nts and nie.
The response of the mutants was characterized using strains of Rhizobium, combined N, and reciprocal grafting (Buttery and Park, 1993). Inoculation of the mutants and wild-type OAC Rico by 18 strains of R. leguminosasarum bv. phaseoli showed some strain x host interaction. R69 had as many nodules as the wild type with all 18 strains but nodules were tiny, pale white and appeared to be nonfixing. R99 showed varying degrees of nodulation ranging from nonnodulation with nine strains (Kim 5s, USDA 2667, 2672, 2672, 2680, CFN42, 227, CE3, and RCR 3622) to several tiny pale nodules with the rest of the strains tested. These results suggest that there may be strain-specificity to the mutant R99. Rhizobium strains had no effect on plant dry weight. With five of the strains tested (USDA 2667, 2668, 2669, 2670, and Lipa Tech strains), acetylene reduction rates were measured at the time of harvest. No activity was detected in R69 and R99 but rates for the wild type varied by strain with a mean of 128 µmol g–1 dry wt. h–1 (Buttery and Park, 1993).
Plant growth response and nitrate reductase activity were tested by N supply (Buttery and Park, 1993). Seed of the two mutants and wild-type OAC Rico was inoculated with turbid broth culture of strain TAL182. Nitrogen treatments consisting of 1, 5, and 15 mM combined N were started 7 d after planting. Plant samples were taken 16 and 29 d after N treatments to assay leaves and roots for in vivo nitrate reductase activity (Buttery and Park, 1993) and to determine nodule number and plant dry weight. At the first sample, the three lines had similar plant dry weight but by the second sample OAC Rico had significantly greater weight than R69 and R99. Nitrogen reductase activity of roots and leaves did not differ between lines, but generally was greater in the high-N treatments. R69 and R99 did not produce nodules. With the wild-type OAC Rico, nodule weights declined as N increased but nodule numbers did not decline significantly.
Reciprocal grafts were made in all combinations between the five lines, the nonnodulating R99; NOD125, a nonnodulating mutant developed by CIAT, Colombia (Pedalino et al., 1992); the ineffective R69; a super-nodulating mutant R32 (Park and Buttery, 1989; Buttery and Park, 1993); and the wild-type OAC Rico when the seedlings were 6 to 10 cm (15–20 d after seeding). The grafted plants were inoculated 2 to 3 d after grafting with a turbid broth culture of TAL182 and supplied with a nutrient solution containing 1 mM combined N. After a further 25 d plants were removed from the pots and nodules were examined. Roots of R99 and NOD125 formed no nodules with any scion. The roots of R69 formed a number of small nodules with all scions, but plant tops remained pale green indicating lack of N fixation. Nodules on the roots OAC Rico and R32 were larger, pink, and presumably effective in N fixation since the plants became dark green. These demonstrated that the nonnodulation and ineffective characters were controlled by the roots and confirmed that the super-nodulation character was controlled by the shoot. The grafting results also agree with epistasic genetic control of the nodulation genes (Buttery and Park, 1993).
It is noteworthy that the nodulation mutants were obtained from a navy bean cultivar adapted to temperate North America so they can be readily utilized in nodulation/N2 fixation studies, particularly in field trials. These nonnodulation/nonfixing mutants may be used as controls to assess available soil N in the measurement of N2 fixation and N assimilation in plants. These mutants have been used to compare plant growth and N accumulation (Shirtliffe et al., 1996) and mycorrhizal activities (Auge et al., 2004).
Like wild-type OAC Rico, both R69 and R99 mutants have green hypocotyls and white flower color, light brown pod at maturity, produce white beans (navy type) with dull seed coat, and have indeterminate growth habit with short vine (IIb).
Seed of R69 and R99 will be maintained at AAFC Greenhouse and Processing Crops Research Centre, Harrow, ON, Canada N0R 1G0. Limited quantities of seed are available for research purposes on request from the corresponding author for the first 5 yr.
ACKNOWLEDGMENTS
We thank R. Armstrong and T. Rupert for their technical assistance.
NOTES
Received for publication October 7, 2005.
REFERENCES
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