32 RESEARCH REPORTS PNL Volume 12 1980
GENETIC VARIATION IN ROOT NODULE FORMATION OF DIFFERENT PEA CULTIVARS
Jacobsen, S. E. and K.W. Henningsen.
Royal Veterinary Agr. Univ., Copenhagen, Denmark
In a search for a crop plant yielding a high level of seed proteins at
a low energy cost, we have analyzed the capacity of some pea cultivars to form
nitrogen-fixing root nodules in the presence or absence of nitrogen and
various strains of the bacterium Rhizobium leguminosarum. The aim of the project
is to elucidate the contribution of the plant and the bacterial genomes in the
establishment and function of root nodules efficient in nitrogen fixation.
The six cultivars of pea listed in Table 1 were studied for their capacity
to develop root nodules in aerated aqueous medium and in vermiculite moistened
with nutrient solution with or without a nitrogen source. The nutrient solution
contained per liter of double deionized water: CaHPO4 lg; K2HPO4 0.2g; MgSO4
0.2g; NaCl 0.2g; FeCl2 O.OOlg; 1 ml of Hutner trace element solution modified
to avoid nitrogen in the medium, and, when required, NH4NO3 0.5g. During
growth, nutrient solution or tap water (a control experiment failed to detect
organisms capable of establishing root nodules) was added to the cultures
to maintain the salt concentration at about the level given above. The plant
cultures were irradiated with 20 (range 9-30) Wm-2 during a 16h photoperiod
and the temperature ranged from 18 to 25C. The seeds were surface sterilized
and germinated in sterile nutrient solution. As the roots developed, some
of the cultures were inoculated with one of 8 known strains of Rhizobium
leguminosarum obtained from the Rothamsted (England) and Uppsala (Sweden)
collections. Prior to inoculation the bacterial cultures were washed and
resuspended in small volumes of plant nutrient solution deficient in nitrogen.
Cultures of the bacterial strains were studied for nitrogen-fixation capacity
as measured by hydrogen evolution and activity of uptake-hydrogenase. The
strains Roth.V, 312, 313, and 1011 were negative with respect to nitrogen
fixation (negative strains), while strain 1008 possessed this activity (posi-
tive strain). The time for nodulation, the size, and the number of nodules
were recorded. The average number of root nodules per plant is listed in
Tables 1 and 2.
It is evident that the pea cultivar 'Monopol', WL 1714 (from the Pisum
Gene Bank, Weibullsholm collection) developed more root nodules than the other
cultivars studied. The cultivar from Jordan Valley, WL 1293, formed some
root nodules when grown on vermiculite (Table 1), but lacked root nodules
completely when grown in aqueous culture (Table 2) in the presence or absence
of nitrogen (N) and/or Rhizobium. Root nodule formation was completely in-
hibited by the presence of fixed nitrogen in aqueous cultures (Table 2).
However, the presence of nitrogen apparently did not influence the number
of root nodules formed by plants developed on vermiculite culture (Table 1).
Plants developed in aqueous culture deficient in fixed nitrogen formed roots
densely covered with root hairs and some nodules were formed by the cultivars
'Dark Skinned Perfection' and Monopol (WL 1714) even when not inoculated with
Rhizobium (Table 2). Inoculation with cultures of Rhizobium leguminosarum
resulted in an increased number of root nodules. The cultivar Monopol
(WL 1714) developed similar numbers of root nodules when inoculated with a
PNL Volume 12 1980
RESEARCH REPORTS 33
nitrogen-fixing strain or any of the non-nitrogen-fixing strains tested.
The cultivar Dark Skinned Perfection appeared to yield more root nodules when
inoculated with the nitrogen-fixing strain.
The cultivar from Jordan Valley, WL 1293, lacking the capacity to form
root nodules in aqueous culture even when inoculated with Rhizobium, has been
reported (1) and (2) to be deficient in the protein lectin (PEA) in the seed.
If lectin is also deficient in the roots of WL 1293, this could be related
to the lack of root nodule formation with any of the Rhizobium strains in
aqueous cultures.
1. Guldager, P. 1978. Theor. Appl. Genet. 53:241-250.
2. Guldager, P., B0g-Hansen, T.C. 1979. Protides of Biological Fluids.
27th F.d. 11. Peeters. pp. 401-404.
Table 1. Average number of root nodules developed per pea plant of different
cultivars grown on vermiculite with nutrient solution. Rhizobium
leguminosarum used for inoculation was strain Roth.V. (The number of
_ plants analyzed for each cultivar is given for each treatment.)
Cultivar or line
Rhizobium
N deficient
present
N added
Rhizobium not added
N added
Fenomen
14
3
11
Dark Skin Perfection
10
10
19
Vinco, WL 1703
19
2
30
Parvus, WL 1107
18
22
1
Monopol, WL 1714
58
72
177
From Jordan Valley WL 1293
18
13
16
Plant per sample
20
20
6
Table 2. Average number of root nodules developed per pea plant of different
cultivars grown in aqueous culture and inoculated with different
strains of Rhizobium leguminosarum. (The number of plants analyzed
_for each cultivar is given for each treatment)
N
deficient
Rhizobium
Cultivar or line
Average of
negative
strains
Positive
strain 1008
present
None
N added
Rhizobium
Present Absent
Dark Skinned
Perfection
101; S=54
160
20
0
0
Monopol, WL 1714
150; S=84
150
53
0
0
From Jordan
Valley, WL 1293
0
0
0
0
0
Plants per sample
100
6
6-12
S = standard deviation