**6. Possiblity of the agronomical use of hypernodulation mutant lines of soybean for promoting yield**

Soybean plants can control the nodule number by auto-regulation of nodulation. Superno‐ dulation or hypernodulation mutant lines of soybean have been isolated from wild type soy‐ bean, and it was expected that these mutants would produce higher seed yield. However, most of these mutants showed reduced vegetative growth and seed yield, possibly due to a heavy burden of excess nodules. Hypernodulation or supernodulation mutant lines of soy‐ beans have been selected from several different cultivars [59-61]. A genetic defect in the au‐ toregulatory control of nodulation causes more profuse nodulation than the wild type (Figure 34). The nodulation trait depends on the shoot genotype and not on the root geno‐ type. In the wild type parents, some shoot-derived signal (autoregulation signal) arrests nodule primordia and suppresses nodule development in response to some signals (infec‐ tion signal) derived from nodulated roots after infection. Sato et al. reported that a rooted single leaf of soybean retains the autoregulation trait.

Recently, the genes were identified for the hypernodulation lines of lotus (*HAR1*) and soybean (*GmNARK*), which play important roles in the autoregulation of nodulation, and they were shown to encode a receptor-like kinase protein that contains a leucine-rich repeat [64-66]. These legume genes are homologous to *Arabidopsis CLAVATA1* (*CLV1*), which is involved in the con‐ trol of cell proliferation in the shoot apical meristem [67]. The results by Ito et al. suggest that the protein coded by GmNARK may play some roles on leaf growth as well.

In spite of profuse nodulation in the hyprnodulation mutant lines, the root and shoot growth is inferior in most of these lines compared with the wild type with or without nitrate supply. All the hypernodulation mutant lines are partially tolerant to NO3 - . The supernodu‐ lation line first reported was nominated "nts", which means "nitrate-tolerant symbiosis" mutant [60]. The labeling experiments using 14CO2 or 13CO2 indicated that the hypernodulat‐ ing mutant NOD1-3 supplied a larger amount of photoassimilate to the nodules than to the roots under nitrogen free conditions, and that the photoassimilate transport to the nodules was less sensitive to nitrate than that of the parent line [69].

Assimilation of 15N2 and 15NO3 was compared among hypernodulation mutant lines, NOD1-3, NOD2-4, and NOD3-7 and the parent Williams [70]. The 5 mM NO3 treatment re‐ sulted in a 95 to 97% decrease in nodule mass and 15N2 fixation by Williams, while the three mutant lines retained 30 to 40% of the nodule mass and 17 to 19% of the 15N2 fixation of con‐ trol Williams. The hypernodulation mutant lines, which had restricted root growth, absor‐ bed less 15NO3 than Williams. These results confirmed that nodule formation and development are less sensitive to external NO3 in mutant lines than in the Williams parent. The partial tolerance of nodulation for nitrate in mutant lines may be partly due to less NO3 absorption activity and smaller roots.

D1, D2, D3, D4 indicate the sampling date (Days after planting) of xylem sap and shoots. We usually sample at R1, R3, R5 and R7 stage for D1, D2, D3, D4. D0 means the planting date. RU %n indicates the relative ureide N percent in xylem sap at sampling time at Dn. RU%n – n+1 means average of RU% at Dn and Dn+1. We use RU%1-0 as RU%1, because we cannot measure

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

**6. Possiblity of the agronomical use of hypernodulation mutant lines of**

Soybean plants can control the nodule number by auto-regulation of nodulation. Superno‐ dulation or hypernodulation mutant lines of soybean have been isolated from wild type soy‐ bean, and it was expected that these mutants would produce higher seed yield. However, most of these mutants showed reduced vegetative growth and seed yield, possibly due to a heavy burden of excess nodules. Hypernodulation or supernodulation mutant lines of soy‐ beans have been selected from several different cultivars [59-61]. A genetic defect in the au‐ toregulatory control of nodulation causes more profuse nodulation than the wild type (Figure 34). The nodulation trait depends on the shoot genotype and not on the root geno‐ type. In the wild type parents, some shoot-derived signal (autoregulation signal) arrests nodule primordia and suppresses nodule development in response to some signals (infec‐ tion signal) derived from nodulated roots after infection. Sato et al. reported that a rooted

Recently, the genes were identified for the hypernodulation lines of lotus (*HAR1*) and soybean (*GmNARK*), which play important roles in the autoregulation of nodulation, and they were shown to encode a receptor-like kinase protein that contains a leucine-rich repeat [64-66]. These legume genes are homologous to *Arabidopsis CLAVATA1* (*CLV1*), which is involved in the con‐ trol of cell proliferation in the shoot apical meristem [67]. The results by Ito et al. suggest that the

In spite of profuse nodulation in the hyprnodulation mutant lines, the root and shoot growth is inferior in most of these lines compared with the wild type with or without nitrate

lation line first reported was nominated "nts", which means "nitrate-tolerant symbiosis" mutant [60]. The labeling experiments using 14CO2 or 13CO2 indicated that the hypernodulat‐ ing mutant NOD1-3 supplied a larger amount of photoassimilate to the nodules than to the roots under nitrogen free conditions, and that the photoassimilate transport to the nodules

sulted in a 95 to 97% decrease in nodule mass and 15N2 fixation by Williams, while the three mutant lines retained 30 to 40% of the nodule mass and 17 to 19% of the 15N2 fixation of con‐ trol Williams. The hypernodulation mutant lines, which had restricted root growth, absor‐

than Williams. These results confirmed that nodule formation and



was compared among hypernodulation mutant lines,

. The supernodu‐

treatment re‐

the RU% at planting.

Relationships

142

**soybean for promoting yield**

single leaf of soybean retains the autoregulation trait.

protein coded by GmNARK may play some roles on leaf growth as well.

was less sensitive to nitrate than that of the parent line [69].


NOD1-3, NOD2-4, and NOD3-7 and the parent Williams [70]. The 5 mM NO3

Assimilation of 15N2 and 15NO3


bed less 15NO3

supply. All the hypernodulation mutant lines are partially tolerant to NO3

**Figure 34.** Nodulated roots of Williams (left) and the hypernodulation mutant NOD1-3 (right).

**Figure 35.** Changes in daily N2 fixation activity and N absorption rate by Williams and the hypernodulation line NOD1-3 cultivated in a sandy dune field in Ikarashi [6].

The hypernodulation mutant lines of soybean may have some advantages for promoting seed yield, due to higher N2 fixation activity or the nitrate tolerant trait to nodulation. Wu

and Harper evaluated the N2 fixation potential and yield of hypernodulating soybean NOD1-3, NOD2-4 and NOD3-7 compared with the parent Williams. In the absence of N fer‐ tilizer, all hypernodulation mutants had greater N2 fixation potential than did Williams in early growth stages. However, the seed yields from the hypernodulation mutants were 10 to 30% less than that for Williams. Suganuma et al. also compared the growth and N2 fixation activity of NOD1-3 and Williams in a sandy dune field. Figure 35 shows the daily rate of N2 fixation and N absorption by Williams and the hypernodulation mutant NOD1-3. The % Ndfa was higher in NOD1-3 (65 %) than Williams (58 %), however, the rates of N2 fixation and N absorption were lower in NOD1-3 than in Williams. The hypernodulation mutant lines have not been used for cultivar improvement, but recently "Sakukei no. 4" bred from En6500 (hypernodulating line from "Enrei") and "Tamahomare" in Japan may be useful in agricultural production by increasing the planting density.
