Abstract
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.
Highlights
Doubled haploids (DH) are an efficient alternative to conventional inbred lines developed through recurrent selfing in maize breeding
Comparison of Doubling Efficiencies in Different Methods of Colchicine Applications In Experiment 1, assessment of success rates associated with three different methods of colchicine application to maize haploids revealed that Method II involving root and crown immersion in
overall success rate (OSR) was about 1.5 times higher in Method II compared to Method I which is commonly used in doubled haploids (DH) production pipelines and comprises of immersing 2–3-day old germinating seedlings
Summary
Doubled haploids (DH) are an efficient alternative to conventional inbred lines developed through recurrent selfing in maize breeding. Compared to conventional inbred lines, DH lines facilitate faster variety registration and intellectual protection due to their distinctness, uniformity, and stability, thereby supporting quicker deployment of maize hybrids in the market [1,2]. The major steps in maize DH line production pipelines are in vivo induction of haploids, separation of haploids from diploids, doubling the genome in haploid (D0 ) plants, and production of D1 seed from D0 plants [3,4]. Plants 2020, 9, 459 large-scale DH line production and continuous process improvement in the last three decades resulted in higher efficiencies at different steps of DH line development [3].
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