Grogg Daniel, Rohner Marius, Yates Steven, Manzanares Chloe, Bull Simon E, Dalton Sue, Bosch Maurice, Studer Bruno, Broggini Giovanni A L
Molecular Plant Breeding, Institute of Agricultural Sciences, ETH Zurich, Universitaetstrasse 2, 8092 Zurich, Switzerland.
Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Plas Gogerddan, Aberystwyth SY23 3EE, UK.
Plants (Basel). 2022 Aug 5;11(15):2054. doi: 10.3390/plants11152054.
Genetic transformation of perennial ryegrass ( L.) is critical for fundamental and translational research in this important grass species. It often relies on -mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. β-glucuronidase (GUS) histochemical staining showed the promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation.
多年生黑麦草（Lolium perenne L.）的遗传转化对于这种重要禾本科植物的基础研究和转化研究至关重要。它通常依赖于农杆菌介导的愈伤组织转化。然而，愈伤组织诱导仅限于对组织培养反应良好的少数基因型。在此，我们报告了从不同多年生黑麦草基因型和外植体（如茎尖、种子和花药）诱导愈伤组织的情况，这些外植体用几种用于功能基因组学的质粒进行了转化。β-葡萄糖醛酸酶（GUS）组织化学染色表明启动子序列在柱头、小穗、花药和叶片中具有活性。我们还用分别允许使用RNA干扰和CRISPR/Cas9进行基因沉默和基因敲除的质粒转化了愈伤组织，目前正在进行基因型和表型研究。使用19种不同的构建体，再生了262个转基因事件。此外，该方案从花药来源的愈伤组织中再生了一个双单倍体转基因事件。这项工作提供了一种概念验证方法，用于扩大适合转化基因型的范围，从而为改善这种重要禾本科作物用于饲料和休闲的研究和育种计划提供服务。
