1. Advancing the production of maize haploids in vivo RESULTS AND DISCUSSION Manifestation of the main inducers’ marker, R1-nj, has been improved in PHI lines (Fig. 3); additionally, they possess a combination of two genes Pl1 and B1 (root and stock marker) allowing haploids to be identified at the stage of 4-day-old seedlings and among mature plants with a rather high accuracy. (Fig. 4, 5 and 6). Improved agronomic performance (plant height, tassel size, logging resistance etc.) was achieved in the PHI lines as well. Based on the previous data, the PHI-2 group was excluded from the study; within the other three groups, PHI-1, PHI-3 and PHI-4, the best lines (F 9 ) were selected and tested in 2013. In crosses with the donor B73 x Mo17, the rate of haploid induction was almost twice as high in the PHI-1 line as in the best initial inducer - MHI (Table 1 and Fig. 7). Segregation studies indicated that there is no relationship between the haploid–inducing ability and male sterility or seed set in the inducers (Rotarenco et al. 2012). In other words, the inducers excelled in high rates of haploid induction (PHI-1) as well as good pollen shedding (Fig. 8) and seed set which also has a very positive influence on the yield of haploid kernels per donor ear (Table 1, Fig. 9, 10 and 11). INTRODUCTION The doubled haploid (DH) technology enables rapid development of completely homozygous lines offering a significant advantage for hybrid breeding. The implementation of DH technology in maize breeding became possible after the creation of specific genotypes with an ability to induce maternal haploids in vivo - “haploid inducers”. However, using this technology in major breeding programs with large numbers of haploids produced from a wide range of donors under different conditions, requires new haploid inducers which combine: (i) a relatively high rate of haploid induction, (ii) a reliable system of marker genes allowing haploids to be identified, (iii) a good pollen production and seed set, and (iv) improved agronomic traits. The objective of our long-term research and breeding project was to create new haploid inducers with the above mentioned requirements. Creation of high-efficient inducers of maternal haploids in the past 10-20 years enabled broad implementation of doubled haploid technology in maize breeding and research. However, using this technology in major breeding programs with large numbers of haploids produced from a wide range of donors under different conditions, requires new inducers which combine: (i) a relatively high rate of haploid induction, (ii) a reliable system of marker genes allowing haploids to be identified, (iii) a good pollen production and seed set, and (iv) improved agronomic traits. Realization of these objectives was accomplished in four new haploid-inducing lines PHI (Procera Haploid Inducers), which were tested during the last three years. The maximal rate of haploid induction in one of the lines (PHI-1) was twice as high (15%) as in the best ancestral inducer - MHI (6-8%). Due to the combination of embryo (R1-nj) and root markers (Pl1 and B1) in the PHI inducers, haploids can be selected at three stages (dry kernels, 4-day old seedlings and mature plants) with a rather high accuracy. There was no negative correlation between haploid-induction ability and pollen production or seed set, which is a required for further improvement of inducers. The most important agronomic traits such as plant height, tassel size and lodging tolerance were significantly improved in PHI inducers. Results from haploid induction experiments of the PHI-3 inducer at Iowa State University (USA) in 2013 were quite comparable with those obtained in Romania. Rotarenco V. 1, Dicu G. 1, Fuia S. 1, State D. 2, Frei U. 3, Lübberstedt T. 3 e-mail: valeriu.rotarenco@procera.rovaleriu.rotarenco@procera.ro MATERIALS AND METHODS Two haploid inducers, Stock 6 (Coe, 1959) and MHI (Chalyk, 1999), were used as initial material. Selection was carried out in the progeny of the hybrid MHI × Stock 6. In 2009, in F 5, there were indentified 4 families (groups) with desirable traits which were named Procera Haploid Inducers (PHI). 56th Annual Maize Genetics Conference, Beijing, China; March 13-16, 2014 1 Procera Genetics, Muncii 47, Fundulea, Calarasi, 915200, Romania, 2 Procera Agrochemicals Ltd, Muncii 47, Fundulea, Calarasi, 915200, Romania, 3 Department of Agronomy, Iowa State University, Agronomy Hall, Ames IA, 50011, USA MHIStock 6PHI-1PHI-2PHI-3PHI-4 The highest haploid-inducing rate (Fig. 1) and the best manifestation of two markers: (1) embryo and endosperm marker (R1-nj gene) and (2) root and stock marker (Pl1 and B1 genes) (Fig. 2, 3, 4, 5 and 6) were the main targets for selection during the next seasons. Fig. 1. The haploid-inducing rates of five inducers in crosses with the donor B73 x Mo17 Haploids Diploids Fig. 5. Selection of haploids in 4-day-old seedlings Fig. 2. The embryo and endosperm marker - the R1-nj gene Stock 6MHI PHI Fig. 6. Pl1 and B1 (stack marker) in the hybrids (B73 x Mo17) x PHIs Haploid inducerMaternal genotype, Donor F1 hybrid HaploidEmbryoless Maternal genotype, Donor Fig. 3. R1-nj in B73 (donor) pollinated with different inducers Fig. 4. The root marker – the Pl1 and B1 genes. Inducer Donor B73Mo17Rf-7B73 x Mo17A619 x B73 Haps/ ear Haps, % Haps/ ear Haps, % Haps/ ear Haps, % Haps/ ear Haps, % Haps/ ear Haps, % PHI-138,718,817,318,322 33,61634,316,2 PHI-37,712,84126196,812,21412,2 PHI-419,510,691012,812,321,68,621,510 MHI20,6137,38,41314,8207,818,310,4 Fig. 7. The haploid-inducing rates of four inducers in crosses with the donor B73 x Mo17 (2013) Table 1. Haploid-inducing rates and the yield of haploids per ear in five donors - three lines (B73, Mo17 and Rf-7) and two hybrids (B73xMo17 and A619xB73) pollinated with four inducers - PHI-1, PHI-3, PHI-4 and MHI (2013) ♂, Self-pollination ♂, PHI-1 ♂, PHI-3 Fig. 9. Female - B73Fig. 10. Female – Mo17 Fig. 11. Female - B73xMo17 DonorTotal seedsHybridsHaploids Haploids per ear Haploids, % Population SA175414453093417,6 Population SP356829456235217,4 The PHI-1 line has been using in haploid production for the Haploid Recurrent Selection to improve two synthetic populations - SA and SP (Table 2 and Fig. 12) (See our poster P199). Fig.12. The ears of the SPC 5 population pollinated with PHI-1 Table 2. The results of haploid induction in two synthetic populations SA and SP pollinated with PHI-1 (2013) ♂, Self- pollination ♂, PHI-1 ♂, PHI-3 ♂, PHI-1 ♂, PHI-4 ♂, PHI-3 It was revealed that the manifestation of the root marker, Pl1 and B1, can be influenced by both the inducer and donor genome; however, it does not affect the quality of haploid identification (Fig. 13, 14 and 15). At the moment, we did not detect any inhibitor genes of this marker, like in case with the R1-nj gene – the C1-I gene (Fig. 16), which makes it a rather reliable tool for haploid screening. Fig. 13. The root marker in B73 pollinated with three inducers – PHI-1, PHI-3 and PHI-4 Fig. 14. The root marker in A619 pollinated with three inducers – PHI-1, PHI-3 and PHI-4 Fig. 15. The root marker in Rf-7 pollinated with three inducers – PHI-1, PHI-3 and PHI-4 Fig. 16. The ear of a donor carrier of the C1-I gene pollinated with a PHI line; identification of haploids by the root marker in 4-day-old seedlings Hybrid inducers, PHI-1 x PHIx3 and PHI-4 x PHI-3, due to their advanced plant height (Fig. 17) and pollen production, are used for haploid induction by open pollination at isolated plots. Their haploid-inducing rates do not differ significantly from the parent lines (Fig. 18). Fig. 17. PHI-4 x PHI-3 Fig. 18. The haploid-inducing rates of two hybrid inducers in crosses with the donors - B73xMo17 and A619xB73 (2013) Conclusions: PHI inducers display a significant improvement in haploid-induction frequency, haploid identification, female and male fertility, and important agronomic traits. These achievements are increasing the efficiency of the in vivo haploid - inducing technology and may extend the application of haploid methods in maize breeding and research. In 2013, the PHI-3 line was estimated at Iowa State University (Department of Agronomy). In general, it had a good performance and the results were quite comparable with those obtained in Romania - the rate of haploids ranged from 8 to 12%, and the seed set, in crosses with donors, was lower in comparison with the other inducers tested at ISU. Fig. 8. PHI-1: Good shedding REFERENCES Chalyk, S.(1999) Creating new haploid-inducing lines of maize. Maize Genetics Cooperation Newsletter, USA, Vol. 73. P.53-54. Coe EH (1959). A line of maize with high haploid frequency. Am. Nat. 93: 381-382. Rotarenco V., Dicu G. (2012) Improvements of in vivo haploid induction in maize. Genetics Cooperation Newsletter, USA, Vol. 86. P.18-21