X-ray-mediated isolated microspore mutagenesis: a system for salt-tolerant line development in wheat (Triticum aestivum L.)

Establishing a technical system for in vitro mutagenesis and salt tolerance screening of wheat microspores is crucial for accelerating the homozygous stabilization of mutant progeny, creating novel salt‑tolerant wheat germplasm, and enabling the effective utilization of saline‑alkali land. Due to technical limitations of in vitro microspore culture in wheat, studies combining wheat microspore culture with in vitro mutagenesis and salt stress screening have not been reported. Based on a previously established genotype‑dependent wheat microspore culture system, our laboratory developed a technical system integrating microspore‑based X‑ray mutagenesis and salt stress screening, thereby providing a new technical approach for accelerating the development of salt‑tolerant wheat germplasm. To determine the optimal X-ray irradiation dose, microspores from the cultivars Shi 4185 and HeNong 6425 were exposed to 0, 3, 5, 10, 15, and 20 Gy of X-ray radiation. The LD₅₀ values for Shi 4185 and HeNong 6425 were 3 Gy and 4 Gy, respectively. To determine the NaCl concentration for in vitro salt tolerance screening of microspores, callus induction and differentiation were carried out on media supplemented with a range of NaCl concentrations. Adding 51 mM NaCl to the induction medium reduced regenerated plants per anther by 62.95% (Shi 4185) and 42.76% (HeNong 6425) relative to controls, while adding 85 mM NaCl to the differentiation medium decreased the regenerated plants by 46.65% (Shi 4185) and 44.02% (HeNong 6425), respectively. Microspores of Shi 4185 and HeNong 6425 were irradiated with X‑rays (3 Gy) and then subjected to salt screening at the induction (51 mM NaCl) and differentiation (85 mM NaCl) stages. Under this dual salt-stress treatment, the regenerated plants per anther of Shi 4185 and HeNong 6425 was reduced by 84.82% and 81.60%, respectively, compared with untreated controls. Overall, 68 and 33 doubled haploid (DH) lines were obtained from Shi 4185 and HeNong 6425, respectively. Through salt tolerance evaluation at the germination, seedling, and 5 dS/m saline‑alkali field stages, 15 salt‑tolerant lines were obtained from Shi 4185 and two from HeNong 6425, with per‑plant yield increases of 56.54%-105.81% and 54.01%-56.07%, respectively, compared with the wild type. Exome sequencing of five salt‑tolerant Shi 4185 DH lines revealed higher numbers of SNP and InDel mutation sites than those in regenerated lines without mutagenesis and salt stress treatment; Under 342 mM NaCl stress, the expression levels of salt tolerance genes TaNHX , TaPDI , and TaMYB in these five lines were significantly higher than those in the wild type Shi 4185. The integrated technical system combining microspore culture, X‑ray irradiation, and in vitro salt stress screening established in this study provides technical support for the development of novel salt‑tolerant wheat germplasm.