Pisum Genetics
2007—Volume 39
Brief Communications
Isolation of pea (Pisum sativum) mutants impaired in arbuscular
mycorrhiza development, using a direct screening
Shtark, O.Y.,1 Ovchinnikova, E.S.,1
Zhukov, V.A.,1 Nemankin, T.A.,1
Titov, V.S.,1 Borisov, A.Y.,1
Gianinazzi-Pearson, V.,2 Seddas, P.,2
Ambrose, M.,3 Ellis, N.3 and Tikhonovich, I.A.1
1All-Russia Res. Inst. for Agri. Microbiol.
St.-Petersburg, Russia
2Mixed Res. Unit Plant-Microbe-Environ.
Dijon, France
3John Innes Centre, Norwich, UK
Arbuscular mycorrhiza (AM) is an ancient symbiotic system formed between 80-90% of terrestrial plants and
fungi belonging to the phylum Glomeromycota. It plays a positive role in plant development due to improved
acquisition of minerals (mainly phosphorus) and water. This beneficial plant-microbe system has a potential for
use in sustainable agriculture. However, the molecular-genetic basis of this symbiosis is insufficiently studied
primarily due to obligatory symbiotic nature of AM-fungi. Until recently, the only approach used for
identification of legume genes controlling AM development was based on the fact that legumes have a common
genetic system responsible both for nitrogen-fixing symbiosis (NFS) and AM. However this approach does not
allow identifying AM-specific genes. In particular, in pea (Pisum sativum L.) eight common symbiotic genes have
been identified using the plant mutants impaired in NFS development (for review see 2). AM-specific pea genes
have not been revealed to date. Only recently, the first three AM-specific legume mutants were identified in
Medicago truncatula using direct screening of a mutagenized plant population (4).
In order to identify similar AM-specific genes in pea, the direct screening of an M2 population of laboratory
line SGE (3) after ethyl-methyl-sulfonate mutagenesis was initiated. A "nurse plant" inoculation system (5) was
specially modified for pea plants and the local conditions, and this system was shown to have several advantages
in comparison with conventional methods of inoculation with AM-fungi. By using the system, more than 700
plants of 193 families were analyzed and 78 putative mutants (PMs) having altered AM phenotypes were
revealed. Thirty-four of them manifested rmc- phenotype (reduced mycorrhizal colonization); twenty-three
displayed pen- (lack of penetration); twelve showed arb (lack or reduced amount of arbuscules) and nine were
myc++ (hypermycorrhizic). Frequency of chlorophyll mutations was about 1.5%.
The seeds of 90% of the chosen plants have been collected and the progeny of PMs have been propagated.
Tests for stability of the revealed phenotypes are now in progress. The progeny of the first plant, the mutant
phenotype of which has been confirmed, is involved in genetic analysis. Also the progeny of twelve PMs was
involved in analysis of symbiotic root nodule formation. The majority of the plants had normal root nodules. The
progeny of a single PM displayed a Nod- phenotype, in that it formed "drumstick" root hairs. It has been shown
that this trait is inherited monogenic and recessively. This genotype has been involved in allelism tests with
different pea mutants in sym8 (1) and sym19 (6) genes, having similar nodulation phenotype.
Thus, the first PMs presumably defective in AM development were isolated in a direct screen. One of the PMs
is probably impaired in the "common" symbiotic gene for both AM and nodule formation. The fact that a
majority of the plants impaired in AM formation had normal nodulation phenotype, indicates, that mutations
have occurred in genes specific to AM development. The genetic analysis of the mutants isolated and confirmed
will permit the identification of new pea genes controlling the development of AM but not NFS.
Acknowledgments: This work was supported by the grants of RFBR (07-04-01171, 07-04-01558, 07-04-13566, 06-04-89000-
NWOC_a), grant of the President of Russia HIII-9744.2006.4, grant of Russian Ministry of Education and Science
02.512.11.2182, grant of Burgundy Administration (07 9201 AA 040 S 3623), NWO grant 047.018.001 and the EC grant
FOOD-CT-2004-506223.
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Pisum Genetics
2007—Volume 39
Brief Communications
1. Borisov, A.Y., Rozov, S.M., Tsyganov, V.E., Kulikova, O.A., Kolycheva, A.N., Yakobi, L.M., Ovtsyna, A.O.
and Tikhonovich, I.A. 1994. Russ. J. Genet. 30: 1284-1292.
2. Borisov, A.Y., Vasil'chikov, A.G., Voroshilova, V.A., Danilova, T.N., Zhernakov, A.I., Zhukov, V.A.,
Koroleva, T.A., Kuznetsova, E.V., Madsen, L., Mofett, M., Nemankin, T.A., Ovchinnikova, E.S., Pavlova,
Z.B., Petrova, N.E., Pinaev, A.G., Radutoiu, S., Rozov, S.M., Rychagova, T.S., Solovov, Topunov, A.F.,
Weeden, N.F., Tsyganov, V.E., Shtark, O.Y., Stougaard, J., Naumkina, T.S. and Tikhonovich, I.A. 2007.
Russian J. of Appl. Biochem. and Microbiol. ("Prikladnaya biokhimiya i mikrobiologiya") 43: 237-243.
3. Kosterin, O.E. and Rozov, S.M. 1993. Pisum Genetics 25: 27-31.
4. March, J.F., Schultze, M. and Oldroyd, G.E.D. 2006. Abstract book of 3rd international conference on legume
genomics and genetics, 9-13 Brisbane, Australia, p 52.
5. Rosewarne, G., Barker, S.L. and Smith, S.E. 1997. Mycol. Res. 101: 966-970.
6. Sagan, M., Huguet, T. and Duc, G. 1994. Plant Sci. 100: 59-70.
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