Development of doubled haploid (DH) plants in wheat is an important technique for establishment of completely homozygous lines in the shortest time frame. In this study, we standardized the in vitro and in vivo variables for the production of wheat DH plants via crossing of three spring wheat genotypes with a wild grass (Imperata cylindrica). The experiment involves testing new treatments and protocol modifications for 2 years. Wheat florets were pollinated with I. cylindrica pollen after 2 days of emasculation, and next day pollinated spikes were injected with different auxin analogues: 100 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or 100 mg/l 3,6-dichloro-2-methoxybenzoic acid (dicamba) or 50 mg/l 2,4-D + 50 mg/l dicamba. A combination of 50 mg/l 2,4-D and 50 mg/l dicamba was superior to other treatments having maximum haploid embryos and haploid plants developed per emasculated florets. The responsiveness of embryo formation to the addition of silver nitrate (AgNO3) (the ethylene inhibitor) with the auxin treatments showed that the presence of silver nitrate in the medium may not be essential for wheat embryo induction. Time of embryo rescue had a significant effect on embryo and haploid plant production. The most successful time for embryo rescue was 24 days after pollination (DAP) resulting in maximum haploid plants developed (40% in wheat variety AKYK92 and 25% each in variety HD2888 and HS542). The immature embryos were cultured in different media, i.e. Murashige and Skoog (MS), half-strength MS, modified MS media, Gamborg’s B5 and modified B5 media. Maximum number of haploid plants were found in half-strength MS media in all accessions (60.0%) followed by modified MS media (40.00%). For colchicine concentration, during 2016–2017, maximum numbers of DHs with less plant mortality were obtained at 0.08 and 0.06% dose. Taking into account the first year results, refinement of the dose in the second year showed that colchicine doses of 0.08 and 0.075% were the best dose for maximizing DH induction in wheat; however, in this case the number of examined plants was very small. Therefore, we can treat the two doses as a guide for further research on larger samples.

中文翻译：

---

不同的体外和体内变量对使用白茅介介导的染色体消除技术在普通小麦中双倍单倍体生产效率的影响

在小麦中培育双单倍体 (DH) 植物是在最短的时间内建立完全纯合系的一项重要技术。在这项研究中，我们通过将三种春小麦基因型与野生草（白茅）杂交，对生产小麦 DH 植物的体外和体内变量进行了标准化。该实验涉及为期 2 年的新治疗和方案修改测试。小麦小花在去雄 2 天后用 I. cylindrica 花粉授粉，第二天向授粉穗注射不同的生长素类似物：100 mg/l 2,4-二氯苯氧乙酸 (2,4-D) 或 100 mg/l 3 ,6-二氯-2-甲氧基苯甲酸（麦草畏）或 50 mg/l 2,4-D + 50 mg/l 麦草畏。50 mg/l 2 的组合，4-D 和 50 mg/l 麦草畏优于其他具有最大单倍体胚胎和每个去势小花发育的单倍体植物的处理。胚形成对添加硝酸银 (AgNO3)（乙烯抑制剂）和生长素处理的响应表明培养基中硝酸银的存在可能不是小麦胚诱导所必需的。胚胎拯救时间对胚胎和单倍体植物的生产有显着影响。胚胎拯救最成功的时间是授粉 (DAP) 后 24 天，导致最大单倍体植物发育（小麦品种 AKYK92 为 40%，品种 HD2888 和 HS542 各为 25%）。未成熟胚胎在不同的培养基中培养，即Murashige 和Skoog (MS)、半强度MS、改良的MS 培养基、Gamborg's B5 和改良的B5 培养基。在所有种质的半强度 MS 培养基中发现了最大数量的单倍体植物 (60.0%)，其次是改良的 MS 培养基 (40.00%)。对于秋水仙碱浓度，在 2016-2017 年期间，在 0.08% 和 0.06% 剂量下获得了最大数量的植物死亡率较低的 DH。考虑到第一年的结果，第二年对剂量的细化表明，0.08% 和 0.075% 的秋水仙碱剂量是最大限度地诱导小麦 DH 的最佳剂量；然而，在这种情况下，受检植物的数量非常少。因此，我们可以将这两个剂量作为对更大样本进一步研究的指导。考虑到第一年的结果，第二年对剂量的细化表明，0.08% 和 0.075% 的秋水仙碱剂量是最大限度地诱导小麦 DH 的最佳剂量；然而，在这种情况下，受检植物的数量非常少。因此，我们可以将这两个剂量作为对更大样本进一步研究的指导。考虑到第一年的结果，第二年对剂量的细化表明，0.08% 和 0.075% 的秋水仙碱剂量是最大限度地诱导小麦 DH 的最佳剂量；然而，在这种情况下，受检植物的数量非常少。因此，我们可以将这两个剂量作为对更大样本进行进一步研究的指导。