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Production and use of doubled haploids (DH) is becoming an essential part of maize breeding programs worldwide as DH lines offer several advantages in line development and evaluation. One of the critical steps in maize DH line production is doubling the chromosomes of in vivo-derived haploids so that naturally sterile haploids become reproductively fertile diploids (DH) to produce seed. This step of artificially doubling the chromosomes is labor-intensive and costly; hence, optimizing protocols to improve the doubling success is critical for achieving efficiencies in the DH production pipelines. Immersion of 3–4-day old germinating haploid seedlings in colchicine solution is commonly used for chromosome doubling in large-scale maize DH line production. This manuscript presents a new method of colchicine application to haploid seedlings that showed superior doubling rates compared to other methods like standard seedling immersion, seed immersion, root immersion, and direct application of colchicine solution to the seedlings at V2 stage in the greenhouse trays. The new method involves immersing the crown region of the haploid seedlings along with all the seedling roots at V2 stage in the colchicine solution. Further experiments to optimize this method indicated that increasing colchicine concentration had a very positive effect on overall success rate in chromosomal doubling, while not drastically affecting survival rate. The optimized method showed on average 5.6 times higher overall success rate (OSR) compared to the standard haploid seedling immersion method which was the second-best method in our experiments. This improved method of colchicine application saves resources by reducing the number of haploids to be generated and handled in a maize DH production pipeline.
In vivo haploid induction in high frequency followed by efficient identification of haploids are important components of deriving completely homozygous doubled haploid (DH) lines in maize. Several genetic marker systems were proposed and/or used for identification of in vivo maternal haploids in maize, such as R1-nj (Navajo), high oil, red root and transgenic markers. In this study, we propose a new method of haploid/diploid identification based on natural differences in seedling traits of haploids and diploids, which can be used in any induction cross independently of the genetic marker systems. Using confirmed haploids and diploids from five different populations, the study established that haploid and diploid seedlings exhibit significant differences for seedling traits, particularly radicle length (RL), coleoptile length (CL), and number of lateral seminal roots (NLSR). In six populations that exhibited complete inhibition of the commonly used R1-nj (Navajo) marker, we could effectively differentiate haploids from diploids by visual inspection of the seedling traits. In the haploid seed fraction identified based on R1-nj marker in ten populations, false positives were reduced several-fold by early identification of haploids at seedling stage using the seedling traits. We propose that seedling traits may be integrated at the haploid identification stage, especially in populations that are not amenable to use of genetic markers, and for improving the efficiency of DH line production by reducing the false positives.
For efficient production of doubled haploid (DH) lines in maize, maternal haploid inducer lines with high haploid induction rate (HIR) and good adaptation to the target environments is an important requirement. In this study, we present second-generation Tropically Adapted Inducer Lines (2GTAILs), developed using marker assisted selection (MAS) for qhir1, a QTL with a significant positive effect on HIR from the crosses between elite tropical maize inbreds and first generation Tropically Adapted Inducers Lines (TAILs). Evaluation of 2GTAILs for HIR and agronomic performance in the tropical and subtropical environments indicated superior performance of 2GTAILs over the TAILs for both HIR and agronomic performance, including plant vigor, delayed flowering, grain yield, and resistance to ear rots. One of the new inducers 2GTAIL006 showed an average HIR of 13.1% which is 48.9% higher than the average HIR of the TAILs. Several other 2GTAILs also showed higher HIR compared to the TAILs. While employing MAS for qhir1 QTL, we observed significant influence of the non-inducer parent on the positive effect of qhir1 QTL on HIR. The non-inducer parents that resulted in highest mean HIR in the early generation qhir1+ families also gave rise to highest numbers of candidate inducers, some of which showed transgressive segregation for HIR. The mean HIR of early generation qhir1+ families involving different non-inducer parents can potentially indicate recipient non-inducer parents that can result in progenies with high HIR. Our study also indicated that the HIR associated traits (endosperm abortion rate, embryo abortion rate, and proportion of haploid plants among the inducer plants) can be used to differentiate inducers vs. non-inducers but are not suitable for differentiating inducers with varying levels of haploid induction rates. We propose here an efficient methodology for developing haploid inducer lines combining MAS for qhir1 with HIR associated traits.
Haploidy Quantitative Trait Loci Maize Phenotypic variation Doubled Haploids Maternal Haploid Inducers Marker-Assisted Selection Phenotyping MAIZE HAPLOIDY ARTIFICIAL SELECTION CIENCIAS AGROPECUARIAS Y BIOTECNOLOGÍA