EVALUATION OF SOME MAIZE INBRED LINES ON FERTILITY RESTORATION PATTERNS OF MALE-STERILE CYTOPLASMS

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1 EVALUATION OF SOME MAIZE INBRED LINES ON FERTILITY RESTORATION PATTERNS OF MALESTERILE CYTOPLASMS Voichiþa HAª *) ABSTRACT The aim of this investigation consisted in detecting the presence of dominant alleles of Rf genes in more than 6 inbred lines provided from the inbreds collection of the Maize Breeding Laboratory, at the Agricultural Research and Development Station Turda. They were crossed with different types of cytoplasmic male sterility: cmsc, cmses, cmsm and cmst. During 992, at the Agricultural Research and Development Station Turda, the observations of pollen restoration reactions were scored. The fertility restoration data clearly showed the relationship between the two representative C and ES of the groupc. Thirtyfour % of the inbreds maintained the male sterility induced by cmsc and cmses, % proved to be restorers and % gave partially fertile plants. Many genotypes (2%) were imperfectly sterile and 33% were partially fertile plants, in cmsm, meaning that for these genotypes, the fertility was easily affected by environment. In the case of restoration reactions of the inbreds in interaction with cmst, only 6% of these, were fully fertile. Generally, the pollen fertility restoration reactions of the inbred lines were in connection with cmssource, cmsversions, of several inbred backgrounds, nuclear x cytoplasmic interaction and environmental conditions. In order to use the cytoplasmic male sterility of different types, in maize hybrid seed production, it is necessary to continue the research for finalizing the cmsanalogues, breeding by backcrossing and selection. Key words: inbreds, cytoplasmic male sterility, pollen fertility restorers. T INTRODUCTION he utilization of cytoplasmic male sterility (cms) in maize hybrid seed production has represented an increasing source of economical efficiency and of improvement of seed genetical purity. The hybrid seed production on the basis of cytoplasmic male sterility imposes to the breeders the task of transformation of maternal forms into male sterile analogues and the paternal ones into pollen fertility restorers. This transformation could be achieved only having genotypes with wellknown reaction versus certain cytoplasmic male sterility types. The complex feature of male sterility cytoplasms and pollen fertility restorer genes interaction imposes the necessity to identify the alleles Rfrf composition at maize inbreds, with a view to their utilization either as maternal genitors or paternal forms (Sarca and Barbu, 982; Cabulea et al., 98; Ciobanu and Partas, 998; Has et al., 22). Nowadays, the most utilized male sterility sources for hybrid seed production are the cmsc and cmss types. In spite of the resistance advantages of the cytoplasmic sources C and S to the bacterial leaf blight incited by the race T of Helminthosporium maydis, certain authors as Duvick and Noble (98), Gracen et al. (99) and Wise et al. (999), have drawn the attention to the danger that in few years, if only the two C and S sources will be utilized, it may be possible to arrive again at the narrowing and vulnerability of the genetic base. For preventing this risk, Duvick and Noble (98), Nemeth (98), Zeng et al. (999) suggest the utilization in maize hybrid seed production of a multiplasm which, by the genetically diversification of cytoplasm, would prevent the unilateral evolution of pest specialized races. After Josephson et al. (98), Darrah and Zuber (986), some USA companies produce hybrid seeds on the basis of cytoplasmic male sterility, of different types, on about 4% from the seed production plot area. The pollen fertility restoration is ensured by the action and interaction of a set of dominant genes. In order to restore the cmst cytoplasm type, the presence of two dominant genes with Rf Rf 2 complementary action as well as the pres *) Agricultural Research and Development Station (A.R.D.S.), 4 Turda, Cluj County, Romania

2 2 ROMANIAN AGRICULTURAL RESEARCH Number 8 / 22 ence of some modifying genes for achieving a complete fertility, is necessary (Duvick, 96; Wise et al., 999). A single Rf 3 dominant gene is necessary for the cmss cytoplasm restoration. The Rf 3 dominant gene expression is strongly influenced by the environmental conditions, conclusion expressed in the papers of the following authors: Duvick and Noble (98), Kheyr Pour et al. (98), Laughnan and GabayLaughnan (983). As regards the genetic control of fertility restoration for the C cytoplasm type, several opinions have been advanced. Josephson et al. (98), KheyrPour et al. (99), KheyrPour and Gracen (98), Laughnan and Gabay Laughnan (983) sustain that for the cmsc fertility restoration type, at least two genes, Rf 4 and Rf genes, would be necessary. Having in view the practical importance of cytoplasmic male sterility and pollen fertility restoration in maize hybrid seed production and the necessity of cytoplasm sources diversification, the aim of this paper was the identification in inbreds genotypes of the recessive or dominant alleles Rf Rf 2 and Rf 4 Rf gene complexes as well as Rf 3 gene with a view to their utilization in genetical transformation programme of parental forms at Turda Agricultural Research and Development Station. MATERIALS AND METHODS As biological material, more than 6 inbred lines and cytoplasmic malesterile analogues, types: cmsc, cmses, cmst, cmsm of seven lines, from the maize inbred lines collection of Maize Breeding Laboratory as part of Turda Agricultural Research Development Station, were utilized. During 99 2, for the identification of the dominant or recessive alleles, of Rf Rf 2, Rf 4 Rf, Rf 3 genes at some inbred lines, the F hybrid progenies, obtained by topcross in single crosses nursery, have been assessed (by degree of pollenfertility restoration). As testers, the male sterileanalogues of some inbreds with identified allelic composition, were utilized; they are also utilized as maternal forms of some perspective hybrids. The F hybrid generations were grown in the observation nursery, on plots of one row of m per each hybrid. According to pollen fertility restoration degree,the inbreds were classified in six groups (after Josephson et al., 98): group I: no anthers extended; group II A: less than half of the anthers extended and all were small, dry and hard, without pollen; group II B: most of anthers extended but all were small, dry and hard without pollen; group III: partially fertile anthers extended with some pollen shed; the proportion of anthers extended was highly variable; group IV (fertility restorers): slightly abnormal anthers with to % extension; group V ( fertility restorers): normal anthers, fully fertile. In the case in which some inbreds manifested an unstable behaviour depending on the environmental conditions or cmstester, their experimentation was repeated during still twothree years. RESULTS AND DISCUSSION In the process of cytoplasmic male sterility utilization in maize breeding programmes, it is as necessary as difficult to identify the inbred lines by the composition of the Rf gene alleles. The identification of the pollen fertility restorer genes at some inbreds consisted of the determination of their reaction to the crossings with male sterile testers cmsc, cmses, cmst, cms M (Table ). In interaction with the C and ES cytoplasms, the pollen fertility restorers identific ation is more complicated, due to the implication of at least two three Rf 4, Rf, Rf 6 complementary genes and some modification factors, probably quantitative ones, which under certain environmental conditions, would act in the absence of Rf gene, influencing the reaction of lines by the appearance of the latebreak phenomenon (KheyrPour et al., 98). Due to the presence of these factors, the observations on lines reaction in C and ES cyto

3 VOICHIÞA HAª: EVALUATION OF SOME MAIZE INBRED LINES ON FERTILITY RESTORATION PATTERNS OF MALESTERILE CYTOPLASMS 3 plasms must be performed in a period of twothree weeks since the emergence of silking. This phenomenon has been recorded in 8% of the inbred lines tested to cmsc, respectively 9% in cmses (Tables 2 and 3). Partas (998). The 24 lines which have proved to be restorers (Table 4) contain in their genotype the Rf 3 dominant allele and they are recommended as genitors in crossing systems or as parental Table. Distribution of lines according to their reaction to different types of cytoplasmic male sterility: C, ES, M, T. No. of cms testerlines No. of tested lines % lines With different reaction Cytoplasm Nonrestorers partially complete Restorers (rfrf) (prf) (Rf) cmsc Total 98 Average cmses Total cmsm Total 2 2 Average cmst Total 223 Average Total tested lines The proportion of nonrestorer genotypes (rf 4, rf, rf 6 ) was of 4% in cmsc, respectively 44% in cmses. As a result of analysing the testcrossings, 6% and 4% of the lines were indentified for completely restoring pollen fertility, which certifies the presence of Rf 4, Rf, homozygousdominant alleles in their genotypes (Tables 2 and 3). The other lines (4, respectively 6%) with partial restoration, include probably, dominant alleles for at least one Rf gene. A number of 2 lines have been tested with the cmsm cytoplasm, out of which only 2% were identified as pollen fertility restorers, which agrees with the results published by Ciobanu and forms. The lines which partially restore the fertility or they have a variable reaction depending on the environ mental conditions, represent 2% of the lines tested on cmsm. This instability is also mentioned in the special literature by Duvick and Noble (98), KheyrPour et al. (98), Laughnan and GabayLaughnan (983). The lines which partially restore the fertility or have an unstable behaviour, are not recommended as parental forms, in reproducing hybrids with improved formula on the basis of cmsm. Concerning the reaction to cmst, 223 inbreds were tested, of which 4% have proved to be nonrestorer (rf, rf 2 ). This fact certifies that the tested lines include only a restorer gene,

4 4 ROMANIAN AGRICULTURAL RESEARCH Number 8 / 22 probably Rf2 gene, more frequent at original inbreds of the Corn Belt (Duvick, 966). Because the cmst is only used in areas less favourable to Helminthosporium maydis Trace, the researches on the use of this cms type are limited (Table ).

5 VOICHIÞA HAª: EVALUATION OF SOME MAIZE INBRED LINES ON FERTILITY RESTORATION PATTERNS OF MALESTERILE CYTOPLASMS Table 2. Testing of restoration ability of cmsc type at some inbred lines at A.R.D.S. Turda, during 99 2 Testerlines Nonrestorer inbred lines (rfrf) Fertility restorer inbred lines (Rf) cms Group I Group II A Group V Group IV W633 cmsc K2, K2B, TC9A, TC22, TB364 TC28 cmsc TA 36 cmsc TC243 cmsc TC84 cmsc TC84, TE23, Lv92, Lv94, Lv3, Lv, TC3, TC3, TB368, TB363, TC36, A66, TC333, TC382 TD6, TD22, TE233, TD269, TC344, TD39, TA4 TD268, TE2, TE2, TC38, TC36, TC383, TC384, TC38, TC39, TC39, TC399, TD34, TD34, TA42, TA432, TA438, F426, MV463, P9, K88, K248C, K2443B, K36A, NF42, NF9, NF228, TD283, TD284, TC396, TC39, TC398, TD32, TD33, TD34, TD3, TD346, TA426, TA42, F43/93, F68/82, F8/86, PT92, PT94, K24 PT94,TD3, TD344 TD298, TE223, TD32, TA49, TA43, K8B, NF32 TD3, TD344, TD348, TA424 PT94 TD23, TD28, TB369, TC322, TC34, TC33, TC33, TC32, TC328, K, K42, K8, P3 TD232, TD236, TD23, TD24, TE28, TE22, TB346, TC33, TC33, TD33, TD336, TD3, TD3, TD32, TD3, TD36, TD3, TD38, TA436, K39B, TD28, TD263, TD29, TD289, TD296, TD29, TD299, TE23, TE26, TE23, TE22, TE226, TE22, TE228, TC339, TC3, TC36, TC36, TC362, TC364, TC368, TC33, TC39, TD329, TD33, TD342, TD34, TD33, TD34, TA43, TA43, TA433, TA43, TA438, TA44, F28, F88/89, F88/89, K63, K9, K96, K8B, K82A, K8C, K, K288, K222, K34A, K346B, NF, NF4, NF9, NF9, NF92, NF93, NF98, NF, TD29, TE2, TC386, TD336, TD346, TD39, TA43, F4/88, PT9, PT98, PT96, PT9, K343, K344, NF4 TD238, A439, TA43 TD28, TE22, TD369, NF3, TD32 Proportion 34% (6/98) %(/93) % (8/98) 4%(8/98) Table 3. Testing of restoration ability of cmses type at some inbred lines at A.R.D.S. Turda, during 99 2 Testerlines cms TC28 cmses TC243 cmses TA36 cmses Nonrestorer inbred lines (rfrf) Fertility restorer inbred lines (Rf) Group I Group II A Group V Group IV TC84, T248B, TC243, TD242, TC28, T24, TB369, TC34, TB36, TB368, TC36, TC3, TC322, TC328, TC33, K42, TC 32 TC3 K, K8, P3, W4 TD28, TD296, TD29, TD298, TD299, TE22, TE23, TE26, TE2, TC383, TC384, TC38, TD298, NF32, TC33, TC38, TD36, NF, TD369, NF 228 NF9 NF4, NF9, NF9, NF3 NF92, NF93, NF98, NF TD6, TE223, TE222, TE22, TC24, TD263, TE28, TE2, TD32, TD348, TE 23,TD34, TD346, TD 348, TE22, TE22, TE226, TE22, TA439, TE22, TA432, TA438, WN, F426/88, TD329, TD33, TD34, TA43, F68/82, PT96 F3/89, F43/93, Lv 86, Lv 92, TA43, F28, F88, F4/88, PT94,

6 6 ROMANIAN AGRICULTURAL RESEARCH Number 8 / 22 Table 4. Testing of restoration ability of cmsm type at some inbred lines at A.R.D.S. Turda, during 99 2 Testerlines cms MKP33 cms M TC 28 cms M Nonrestorer inbred lines (rfrf) Fertility restorer inbred lines (Rf) Group I Group II A Group II B Group V Group IV TC24, TE2, TB329, TB3, K TC9B, TC4, TC84, T243, T248, TD22, TB362, TB366, TC32, TC328, TC33, TC33, TC384, TC38, TA49, K, P, PT96, ND24 T248, T29, TC24, TB362, TB366, TB3, P,W3R,W 633, A66, ND24 T29, TC243, TC24, TD24, TB364, TB3, TC33, TA44, TA432, K348A, CO26 TC29, TC243, TA36, TA44, K9, W, CM A66, TC29, K8, PT9 Precoce46, CM2, CO26, CO2, A66, K26 T248B, TC242, TD233, TD283, TE2, TB363, TB36, TB369, TC386, TC39, TC39, TC396, TC39, TD33, TA424, TA428, TA436, PT98 T69a, TC28, TC4 TD6, TC28, TD233, TD284, TD29, TE28, TA36, TC34, TC344, TC39, TC399, TD3, TD32, TD39, TA42, TA426, TA42, TA429, TA433, TA43, K42, K, K8B, K2, K346B, PT94, PT39, PT9, PT9, PT92, PT93, PT94, PT9, PT9, A66, CM2, CO2 Proportion 2 % (24/2) 8 % (22/2) 9 % (/2) 2 % (24/2) 33 % (4/2) Distribution of inbreds in groups on the basis of fertility restoration, performed by Josephson et al. (98): Group I no anthers extended Group II A less than /2 of anthers extended, but they are small, dry and hard, without pollen. Group II B most of anthers extended, but they are small, dry and hard, without pollen. Group III partially fertile anthers extended with some pollen shed. Proportion of anthers extended was highly variable. Group IV slightly abnormal anthers with % extension. Group V normal and completely fertile anthers. Table. Testing of restoration ability of cmst type at some inbred lines at A.R.D.S. Turda, during 99 2 Tester lines Nonrestorer inbred lines (rfrf) Fertility restorer inbred lines (Rf) cms Group I Group II A Group V Group IV W633 cms T TC84, TC22, K2 K2B TC9A, TC28 cms T TC82, TC246, TD232, TD233, TD23, TD236, TD242, TB36, TB368, TB369, TC33, TC34, TC322, TC32, TC328, TC33, TC333, TC33, TC344, TA46, K42, K, K8, K23, P22, P3, CO2, W4, A66, A66 TC3, TB33, TC36, TC36, TC364 TC3 TC243 cms T TD34, TC394, TD39, TD33 TD268, TD26, TD28, TD296, TD29, TD298, TD299, TE2, TE23, TE26, TE233, TC33, TC3, TC36, TC362, TC36, TC368, TC38, TA36 cms T TC38, TC38, TC383, TC384, TC38, TD336, TD343, TD344, TD34, TD3, TD3, TD32, TD33, TD3, TD36, TD3, TD38, TD39, TD36, TD36, TA49, TA42, TA43, TA433, TA44, P9, MV463, PT9, PT92, PT93, PT94, PT9, PT96, K93, K, K9, K96, K8B, K82A, K8C, K86, K248C, K224, K238, K2443B, K34, K36B, NF, NF4, NF32, NF4, NF42, NF6, NF9, NF9, NF92, NF98, NF3, NF, NF228, NF288 TD29, TE22, TE22, K224, TC384 TC326, TD39, TE2, TC39, TC39, TC399, TD28, TD289, K63 TC 28 cmst TC28, TD284, TE28, TE223, TC39, TC386, TD3, TA426, TA428, TA429, TA43, TA432, TA43, PT9, PT93, PT94, PT9, PT9, PT98, PT9, K343, K344, K346A TC39, TC39, TC399, TD32, TD33, NF228 TC33 cms T TC24, TE2, TE22, TE222, TE22, TE226, TE22, TD32, TD329, TD33, TD34, TD34, TD346, TD34, TD348, TD34, WN, F426/88, F9/86, F88/89, F88/89, F3/89, F68/82, K24 TE22, TE223, TE22, TE226, TE22, F8/86, F9/86 TD263, TD28, TD289, TC36, F28, TD29, TD28, TD289, TD22, TE22, TC84 cms T TD342, K8B, K36A, K39B, PT9, PT9, TE22, TE23, TE23, PT96, PT9 TD33, TE229 TE228, Proportion 4 % (6 / 223) 9%(2/223) 6 % (3 / 223) % (2/223)

7 VOICHIÞA HAª: EVALUATION OF SOME MAIZE INBRED LINES ON FERTILITY RESTORATION PATTERNS OF MALESTERILE CYTOPLASMS A special behaviour has been noticed on the inbred lines T 69a and TD 34 (Table 6). Table 6. Behaviour of some inbreds with different testers cms Testerlines cms S 42 cms T W 33 cms T T 3 cms T T 248 cms T A 28 cms T TA 36 cms T TC 33 cms T TC 243 cms T Nonrestorer inbred lines (rfrf) T 69a T 69a T 69a TD 34 TD 34 Fertility restorer inbred lines (Rf) T 69a T 69a TD 34 They presented a different reaction of maintenance or restoration of pollen fertility in relation to the maternal genotype. This fact is explained by the presence of Rf gene in the male sterility genotype analogues of lines S 42 cmst, A 28 cms T, TC 243 cmst respectively, which in interaction with the complementary gene Rf2 (more frequently in the genotype of some inbreds) into the genotype of T 69a, TD 34 inbreds, achieves the complete restoration of cmst type. This more special behaviour of these two inbreds sustains the necessity to verify the inbreds reaction on both many male sterility types and different cms genotypes. This conclusion was encouraged by Duvick and Noble (98) too, who underlined the importance which should be given to the choosing of some cms testers with a clear reaction, no matter of nuclearcytoplasmic interaction or environmental conditions. CONCLUSIONS The behaviour of different lines towards male sterility has manifested as a specific feature dependent on the used male sterility source, genotype of cmsanalogue, specific character of the nuclearcytoplasmic interaction and environmental conditions. The frequency of lines with dominant Rf genes manifested for the cmsc and the cms ES was of %, respectively %, in comparison with the proportion of only 6% lines, in the genom of which the dominant genes with complementary action Rf Rf 2 useful for cmst restauration are present. The verification of inbreds to cmsm(s) type emphasized the high proportion (33%) of lines with an unstable behaviour; therefore these lines should not be recommended as parental forms for improved hybrids releasing. The T 248, TB 36, A 66 inbred lines have been noticed only by the restoration ability of cmsm and the maintainer cmst, cmsc and cmses. Therefore, these lines could be utilized as indicators of cmsm type. The observations performed on the restoration reaction of cmsc and cmses demonstrate the close relationship between the two representatives of C group. REFERENCES Cãbulea, I., Haº, V. Haº, I., Grecu, C., 98. Unele aspecte privind utilizarea androsterilitatii citoplasmatice la porumb. Contributii ale cercetarii stiintifice la dezvoltarea agriculturii. Volum omagial: Ciobanu, V. Gh., Partas, E., 998. Identificarea alelelor genelor Rf la liniile de porumb. Cercetari de genetica vegetala si animala, vol.v: 63. Darrah, L.L., Zuber, M.S., 986. United States farm maize germplasm base and commercial breeding strategies. Crop Science, vol.26: 9 4. Duvick, D.N., 96. Cytoplasmic pollen sterility in corn. Adv. Genetics 3: 6. Duvick, D.N., 966. Influence of morphology and sterility on breeding methodology. Plant breeding. A symposium held at Iowa State University. The Iowa State University Press, Ames, Iowa: Duvick, D.N., Noble, S.W., 98. Current and future use of cytoplasmic male sterility for hybrid seed production. Maize Breeding and Genetics (ed. D.B. Walden): Gracen, V.E., KheyrPour, A., Earle, E.D., Gregory, P., 99. Cytoplasmic inheritance of male sterility and pest resistance. Proceeding of the 34 th Annual Corn and Sorghum Conference. Haº, V., Haº, I, Grecu, C., 22. The use of cytoplasmic malesterility in maize seed production. VII th Congress of the European Society for Agronomy, Cordoba, Spain, Proc.: Josephson, L.M., Morgan, T.E., Arnold, J.M., 98. Genetics and inheritance of fertility restoration of malesterile cytoplasms in corn. Proceeding of the 33, Annual Corn and Sorghum Research Conference, Chicago. KheyrPour, A., Gracen, V.E., Everett, H.L., 99. Genetics of cmsc fertility restoration. Maize genetics cooperation Newsletter, Nr.3.

8 8 ROMANIAN AGRICULTURAL RESEARCH Number 8 / 22 KheyrPour, A., Gracen, V.E., 98. Genetics of cmsc fertility restoration. Maize Genet. Crop News 4: 9. KheyrPour, A., Gracen, V.E., Everett, H.L., 98. Genetics of fertility restoration the Cgroup of cytoplasmic male sterility in maize. Genetics, vol. 9: Laughnan, J.R., GabayLaughnan, S., 983. Cytoplasmic male sterility in maize. Am. Rew. Genet. : 248. Nemeth, I., 98. Broading the genetic base for maize breeding in Europe. Congress of the Eucarpia Maize and Sorghum Section, Montreux. Sarca, V., Barbu, V., 982. Cercetari privind folosirea androsterilitatii citoplasmatice de tip C si El Salvador, în producerea unor hibrizi de porumb. Probl. genet. teor. aplic., XIV (4): Wise, R.P., GobelmanWerner, K., Pei, D., Dill, C.L., Schnable, P.S., 999. Mitochondrial transcript processing and restoration of male fertility in T cytoplasm maize. Journal of Heredity 9(3). Zeng, M., Liu, S., Yang, T., Liu, Y., Li, S Breeding and genetic analysis on the multiplasmic lines of maize. Maize genetics cooperation Newsleter 2:.

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