A high incidence of the sources of cytoplasmic male sterility (cms) in the Maize Research Institute (MRI) gene bank

--Vancetovic, J, Vidakovic, M, Vidakovic, M, Rosulj, M

Finding a restorer cytoplasm, which exists only theoretically, as proposed by Hermsen (Euphytica 14:221–224, 1965; Euphytica Supp. 1:63–67, 1968), would allow easier and genetically cleaner hybrid seed production based on male sterility in a plant species than any other system imposed so far. Only a limited amount of research has been done in this area, and all of the results were negative (Kohel and Richmond, Crop Sci. 3:361–362, 1963, on cotton; Rutger and Jensen, Euphytica 16:350–353, 1967, on barley; and Washnok, MNL46:25–27, 1972, on maize). This encouraged us to start a huge experiment, and search all of our gene bank for the presence of the restorer cytoplasm for the gene ms10. Unfortunately, we have not found it. So the objective of the original study was quite different from the results reported herein, not to find new sources of cms, but to find the restoring cytoplasm for the gene ms10 in maize.

In a search for the restorer cytoplasm (Vidakovic et al., J. Hered. 93:444–447, 2002) for the gene ms10, each accession from the MRI gene bank was crossed with a heterozygous Ms10/ms10 tester. The plants from the two crossed ears of each heterogeneous accession (OP varieties, races, synthetics), and from one ear of the crossed lines, were subsequently backcrossed with the Ms10/ms10 tester. Apart from the Ms10/ms10 genetic constitution, the heterogeneous tester consisted of various genotypes from different maturity groups, in order to cover almost all of the vegetation span of the gene bank accessions. Crossing and backcrossing was done in isolation, with detasselled gene bank accessions used as females. Twenty plants of the first cross per each ear were used for backcrossing within each accession. Approximately the same number of kernels from the backcrossed ears were mixed within an accession, and subsequently planted in a population of about 60 plants for testing for the presence of the restoring cytoplasm for the ms10 gene. Indication of the presence of the restoring cytoplasm would be a 100% fertile test, and average expected frequency of the sterile ms10/ms10 plants was 1/8 for each accession.

Restorer cytoplasm wasn’t found, but a high incidence of the sources of cytoplasmic male sterility was obvious within our gene bank. Since plants within each test were descendants from a single ear, they all shared the same cytoplasm. As an indication that an accession contains a sterile cytoplasm we took the presence of more than 30% sterile plants in the test, which is significantly higher than the theoretical 1/8 which would arise from ms10/ms10 plants only. In Table 1 there is a list of accessions where sterile cytoplasm was indicated.

In the year 2000, spare seed from the crosses and/or backcrosses of these accessions was used for multiplication by open-pollination (cytoplasm is only transmitted through female plants). Genotypes with more than 40-50% sterile plants in the phase of multiplication (from the original seed lots) were chosen for testing of the type of cytoplasm involved (± in Table 1).

Some more sources of sterile cytoplasms were indicated from an unexpected source — from so-called test-candidates for the restoring cytoplasm, which were 100% fertile in the test. Within those tests, selfing of individual plants and outcrossing to the ms10/ms10 tester was performed. A 100% fertile self plus 100% sterile or 1:1 (Ft:St) segregating test would indicate the presence of the restorer cytoplasm. Such pairs of selfs-tests we did not find, but in some of the selfed progenies an excess of sterile plants was observed, far greater than 0.25, which would be expected if the original plant was heterozygous for the ms10 gene. These progenies were also included in the multiplication field in 2000, and few were (Table 2) chosen for the test of the type of cytoplasmic sterility (based, again, on more than 40–50% sterile plants in the phase of multiplication).

In the multiplication field, progenies were also included from the ears of the sterile plants found in the accessions of the Yugoslav Collection, taken from the open-pollination. Again, for further classification, only progenies with more than 40% sterile plants were chosen, indicating the presence of the sterile cytoplasm, and not solely some of the ms genes. These accessions are listed in Table 3.

Kol 146 will be included twice in the classification test (Table 1 and 3), chosen by the 2 criteria (descendants are from the 2 different ears from this variety).

The high incidence of sterile cytoplasms in our gene bank is somewhat surprising. We have chosen 50 sources for a test of the type of cytoplasmic male sterility, but it seems that the number of the cms sources is at least twice that high, giving a number of about 100 independent sources of cms. In comparison with the total active number (about 5000) of accessions in our gene bank (accessions that are actually present), this gives a total of 2%. It raises a question of whether there may be any evolutionary significance of a high number of, mostly, populations with cytoplasmic male sterility, possibly in making some self-compatibility barrier protecting such a population from foreign pollen not carrying the appropriate restorer (Rf) gene.

Sterile plants from the accessions chosen are crossed for further classification in 2002 with 3 heterozygous testers: RfT/rfT, RfC/rfC and RfS/rfS. Field classification for these 3 types of cms will be done in 2004, since our breeding nursery in 2003 was destroyed by a storm.

We would like to thank our technical staff, Stanija Mladenovic and Snezana Veselinovic, and our workers Milijana Kalisanin and Milena Petrovic, for performing a huge amount of work in the laboratory and in the field.


Table 1. Accessions with more than 30% sterile plants in an ms10 restorer cytoplasm test, indicating the presence of the sterile cytoplasm.

Kol-Int(-l)† Acc.no Name of accession Provenience LB‡ Remark
Kol 36 Domaci bijeli Orahovo LB +  
Kol 146 Zuti tvrdunac Sipovo   +  
Kol 239 Mnogoredi zuti zuban     +
Kol 420 Zuti tvrdunac Kabas LB    
Kol 536 Zlatni zuban Prislonica   +
Kol 1041 Zuti poluzuban Prosenikovo LB    
Kol 1084 10-redi Zeta LB    
Kol 1232 Saradan rani Boljevic LB  
Kol 1299 Srednje seme Bjelo Polje LB    
Kol 1326 Klek Prigradna   +  
Kol 1385 Domaci Goles   +  
Kol 1393 Domaci Spajici LB    
Kol 1415 Domaci bijeli Bogomilovici      
Kol 1416 Domaci bijeli Pjesivci LB    
Kol 1436 Domaci Sinj LB    
Kol 1575 Bosanac Zenica LB    
Kol 1609 Beli Kalna LB    
Kol 1729 Poljski osmak Visegrad LB    
Kol 1922 Domaci slatki Sinj      
Kol 1944 Domaci tvrdunac Sipovo   +  
Kol 1947 Cado Sipovo   +  
Kol 2170 Domaca bakrena trdinka Radovljica LB    
INT 2975 Mestnaja kavkazkaja zeltaja Kavkaz LB    
INT 3497 Nebraska long ear Xchalq-composite Mexico   + #
INT 3506 Amarillo bajio Mexico   +  
INT 3635 Korom abad 3 Iran     #
INT 3727 Cuarentin 1938×45 Argentina   + ††
INT 3730 Cuarentin 1939×35 Argentina   + ††
INT 3732 Cuarentin 1938×33 Argentina     ††
INT 3734 Cuarentin 1932×45 Argentina   + ††
INT 3735 Cuarentin 1933×39 Argentina   + ††
INT 3938 XIX/44 Synthetic USA   +  
INT 4581 Jordi Jordan   +  
INT 4961 Hasuri Gruzia   +
INT 4965 Ahmeta S. Birkiani Gruzia      
INT 5012 Kremnistaja belaja Gruzia      
INT 5267 PD 1109 population DDR      
INT 5283 PD 1156 population DDR   +
INT 5307 PD 1302 population DDR   +
INT 5313 PD 1416 population DDR   +  
INT 5399 Brzovec Bulgaria LB    
INT 6100 Voronjezskij M 52 synth. Former SSSR   +  
INT 6651 Pool 42 (NTR-2) Mexico      
INT 7106 MG 91 862 population Bari g.bank-Italy   + #
INT 7154 MG 91 912 population Bari g.bank-Italy LB    
INT 7224 MG 91 774 population Bari g.bank-Italy LB    
INT-L 1472 R-563 Bari g.bank-Italy LB    
INT-L 4884 GR.38868 Greece   +  
INT-L 5771 WCB-27 Greece   +  
INT-L 5856 Rt 1 Tchecoslovachia   +  
INT-L 5857 CE-178Rf Tchecoslovachia   +  
INT-L 5860 RT 2 Tchecoslovachia LB +  
INT-L 6136 Rt 11 Tchecoslovachia LB +  
INT-L 6137 Rt 25 Tchecoslovachia LB +  
INT-L 6276 SR 10 (flint) Poland   +  

† Kol=Collection of OP varieties from the territory of the former Yugoslavia
INT=Heterozygous foreign material
INT-L=Foreign and domestic inbred lines
‡ LB=Sterile plants in the test (some or all) exhibited the late break of sterility
§ +=Chosen for the sterility type classification
¶ = 1 of 2 progenies gave 100% St test
# = The test was almost 100% sterile
††= Originally received as segregating for St plants


Table 2. Selfed candidates for the restoring cytoplasm chosen by the excess of sterile plants in the selfed progenies (significantly greater than 0.25).

Kol-Int(-l) † Acc.no Name of accession Provenience +‡
Kol 237 Zuti zuban from sister crosses Zemun Polje +
Kol 393 Belo staro seme Rznic +
Kol 547 Krupni staklarac Vica +
Kol 842 Crveni kukuruz Jastrebarsko +
Kol 1387 Domaci bosanski Bielo Bucje +
Kol 2154 Domaći zuti Prilep +
INT 5019 Ambrolauri S. Nikorcminda popul. Gruzia +
INT 7106 MG 91 862 population Bari g.bank-Italy +
INT-L 5767 W 182B Greece +
INT-L 5981 SV 59 Tchecoslovachia +

† Kol=Collection of OP varieties from the territory of the former Yugoslavia
INT=Heterozygous foreign material
INT-L=Foreign and domestic inbred lines
‡ +=Chosen for the sterility type classification


Table 3. Progenies chosen for sterility classification from the sterile plants from the open-pollination of the accessions of the Yugoslav Collection, based on an excess of more than 40-50% sterile plants during multiplication.

Kol Acc.no Name of accession Provenience +†
Kol 146 Zuti tvrdunac Sipovo +
Kol 326 Zuti tvrdunac Novi Sad +
Kol 1127 RB 10 Var. vulgata Bitolsko +
Kol 1172 RB 55 Var. vulgata Probistip +
Kol 1258 Domaci beli Crmnica +
Kol 1385 Domaci beli Goles +
Kol 1613 Beli Svrljig +
Kol 1882 Domaci D 1597 Gracac +
Kol 2100 Bosanski zutac Staro Sipovo +

† +=Chosen for the sterility type classification