Leveraging natural climatic advantages for large‑scale wheat doubled haploid production via wheat × maize: a protocol...

Doubled haploid (DH) lines generated via the wheat × maize system represent an effective technology for accelerating wheat breeding. However, achieving low-cost, large-scale DH production remains a core technical challenge.

In this study, we conducted flowering-synchronization experiments to determine the hybridization window between wheat and maize. Wheat was sown year-round in the field, while maize was planted both in the field and in high tunnels. We also evaluated a novel pollination method, four new nutrition solutions for cut-tiller culture, and a newly designed optical device for screening embryo-containing caryopses prior to embryo rescue. The results are as follows: (i) spring wheat, as well as vernalized facultative and winter wheat can be naturally grown year-round in Kunming, a plateau region in China with a mild climate. The hybridization window between wheat and maize is five months (June-October) in natural conditions, but can be extended to 10 months (April-next January) through high-tunnel maize cultivation in January-February and August-September; (ii) compared with commonly used brushing pollen pollination, the rolling pollination method increased pollination efficiency more than tenfold, and raised the haploid embryo formation frequency (EFF) by 1.9 times; (iii) one new nutrient solution (A) for cut-tiller culture performed significantly better than the currently used nutrient solution B. It achieved the highest EFF (46.39%), caryopsis setting rate (97.20%), and 1000-caryopsis weight (28.49 g) among five solutions. Solution A, without sulfurous acid and silver nitrate (both present in solution B), is also more cost-effective and environmentally friendly; (iv) pre-screening caryopses using a newly designed optical device prior to embryo rescue demonstrated a 98.6% accuracy rate and a 3.46% false-negative rate in selecting embryo-containing caryopses. This method doubles the efficiency of embryo rescue and remarkably reduces the use of sterilants and labor in mass DH production. The optimized protocols were validated from 2022 to 2024, yielding over 137,080 DH lines from 1,039 diverse wheat materials.

The optimized protocol significantly improved doubled haploid production efficiency and reduced its cost, providing a more efficient and cost-effective system for mass production of wheat doubled haploids via wheat × maize. It is expected that the optimized protocol for wheat DH production will be applicable in regions with similar climates.