Cellular Differentiation and Molecular Genetics Section, Department of Botany, Jamia Hamdard, New Delhi 110062, India.
Genomic and Biotechnology Unit, Department of Biology, Faculty of Science, University of Tabuk, Tabuk 71491, Saudi Arabia.
Genes (Basel). 2022 Dec 11;13(12):2335. doi: 10.3390/genes13122335.
L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85-90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%-95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.
L. 是一种具有重要治疗作用的植物，可合成重要的强心苷，如洋地黄毒苷和地高辛。本工作报道了一种详细而有效的繁殖方案，通过优化 Murashige 和 Skoog (MS) 培养基中的各种 PGR 浓度来繁殖 。通过流式细胞术（FCM）和起始密码子靶向（SCoT）标记技术评估了体外再生体的遗传同质性。首先，将接种在完全 MS 培养基中添加 0.5mg/L GA 的种子接种在幼苗中。使用下胚轴、节、叶和顶芽等不同部位作为外植体。单独或与 2,4-D/NAA 组合使用 BAP 可获得致密的愈伤组织。下胚轴衍生的愈伤组织诱导体细胞胚，在 0.75mg/L 强化 BAP 的 MS 培养基中增殖和萌发最好。扫描电子显微镜（SEM）图像证实了存在各种体细胞胚的发育阶段。获得了芽再生，其中 1.0mg/L BAP 和 2.0mg/L BAP+0.5mg/L 2,4-D 被证明是诱导直接和间接芽芽的最佳 PGR 处理。再生芽在 1.0mg/L IBA 增强培养基中具有最高的生根率（87.5%），每个芽具有 24.7±1.9 条根。生根的组培苗在温室中以 85-90%的存活率适应。田间生长、体细胞胚再生和器官发生衍生植物的基因组大小和 2C 核 DNA 含量分别估计为 3.1、3.2 和 3.0 皮克（pg）；没有倍性状态和 DNA 含量的改变，验证了遗传均匀性。6 个 SCoT 引物揭示了所有分析的植物样本中 94.3%-95.13%的单态带，进一步表明了体外克隆和母株之间的遗传稳定性。本研究首次成功地从下胚轴愈伤组织诱导体细胞胚；流式细胞术和 SCoT 标记证实了再生植物的遗传同质性。
