Sorghum is considered a challenging species for transformation due to the limited choice in genotypes that can be transformed and the reliance on callus derived from immature embryos that need to be teased out of developing seeds. We have developed a more practical tissue culture procedure to generate embryogenic callus from leaf whorl explants obtained from 4 week-old seedlings. This procedure offers greater ease of obtaining explants, enables a more rapid turnover of growth chamber or greenhouse plants that can be used for explant collection, and eliminates the dependency on obtaining flowering plants with adequate numbers of seeds. Callus derived from these somatic embryos was amenable to biolistic transformation with DNA-coated gold particles and transformants were shown to express the Cas9 transgene. A comparison between genotypes RTx430 and P898012 indicated that the former was more suitable to tissue culture from leaf whorls based on greater callus induction and regeneration. Callus from both genotypes secreted colored compounds into the medium that we determined to be phenolic in nature, likely 3-deoxyanthocyanidins, based on UV–Vis absorbance profiles and thin layer chromatography. In order to reduce negative impacts of these compounds on regeneration, the addition of activated charcoal (AC) or polyvinylpyrrolidone (PVP) to the culture medium was examined. The addition of 0.5 g L⁻¹ of AC after 4 weeks on callus induction medium accelerated callus regeneration, whereas culturing leaf whorl explants on medium containing 1 g L⁻¹ PVP reduced the production of colored compounds and enhanced callus growth.

高粱被认为是转化的挑战性物种，因为可以转化的基因型选择有限，并且依赖于需要从发育中的种子中摘除的未成熟胚衍生的愈伤组织。我们已经开发了一种更实用的组织培养程序，可以从4周龄幼苗获得的叶片螺纹外植体中生成胚性愈伤组织。该程序使获得外植体更加容易，使可用于外植体收集的生长室或温室植物的周转速度更快，并且消除了对获得具有足够数量种子的开花植物的依赖。来自这些体细胞胚的愈伤组织适合于用涂有DNA的金颗粒进行生物弹射性转化，并且显示了转化子表达Cas9转基因。基因型RTx430和P898012之间的比较表明，前者基于愈伤组织的诱导和再生，更适合于叶轮组织培养。两种基因型的愈伤组织都会根据UV-Vis吸收曲线和薄层色谱，将有色化合物分泌到我们确定为酚类的培养基中，可能是3-deoxyanthocyanidins。为了减少这些化合物对再生的负面影响，研究了向培养基中添加活性炭（AC）或聚乙烯吡咯烷酮（PVP）的方法。4周后在愈伤组织诱导培养基上添加0.5 g L ^-1的AC可加速愈伤组织的再生，而在含1 g L ^-1的培养基上培养叶轮外植体 PVP减少了有色化合物的产生并增强了愈伤组织的生长。