A Study on the Integration of In Vitro Methods with In Vivo Double Haploid Technique in Maize (Zea mays L.)

Abstract

The in vivo doubled haploid technique in maize breeding significantly reduces the time required for developing homozygous lines, offering advantages in terms of both time and cost. Although this technique enables the development of lines much faster than traditional breeding methods, ongoing research aims to further shorten the development process through alternative approaches. In this context, significant efforts have been devoted to integrating in vitro methods with in vivo doubled haploid technique. This study aimed to investigate the potential of combining in vivo and in vitro techniques for the rapid development of homozygous maize lines. A total of 10 local populations and 3 inducer lines (CIM2GTAIL-P2, ADAIL-1, STOCK-6) were used as experimental material. The study was conducted in two phases under field and laboratory conditions. During the first phase, induction crosses were performed in 2022, and the haploid induction rates of donor genotypes were found to range from 1.29% to 7.12%, as determined using the Navajo marker. In the laboratory phase, immature embryo culture was employed for both direct and indirect regeneration using samples collected 18–20 days after induction crossing. Haploid status of the samples obtained through direct regeneration was confirmed using the Feulgen chromosome staining method. Four of the donor materials (DON3, DON4, DON6, DON7) yielded successful results in tissue culture studies. Explants were taken from immature embryos to CHU medium for callus formation and then these calli were transferred to Murashige and Skoog medium for the formation of somatic embryos. This approach enabled the production of 3 to 6 calluses per immature embryo, depending on the donor genotype. The results of this study indicate that integrating immature embryo culture as an in vitro method into the in vivo doubled haploid technique can offer benefits in terms of both time efficiency and an increased number of developed materials.