Qahtan Ahmed A, Faisal Mohamad, Alatar Abdulrahman A, Abdel-Salam Eslam M
Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.
Plants (Basel). 2021 Dec 20;10(12):2820. doi: 10.3390/plants10122820.
L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of . Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.
L.是一种柑橘科常绿灌木，以其精油和多种生物活性而闻名于世，显示出其潜在的药用价值。在本研究中，我们调查了不同培养条件，包括植物生长调节剂、培养基类型、培养基pH值和碳源，对L.茎段外植体离体再生的影响。培养8周后，在pH值为5.8的Murashige和Skoog（MS）培养基中，添加3.0%蔗糖、5.0 μM 6-苄基腺嘌呤（BA）和1.0 μM 1-萘乙酸（NAA），获得了最高的再生率（96.3%）和最多的芽数（40.3个芽/外植体），芽长为4.8厘米。为了生根，将单独收获的小芽转移到添加了吲哚-3-乙酸（IAA）、吲哚-3-丁酸（IBA）或NAA的1/2 MS（半强度）培养基上，6周后，0.5 μM IBA在根诱导（91.6%）、根数（5.3条）和根平均长度（4.9厘米）方面取得了最佳效果。平均95.2%的健康离体再生植株在移植到盆栽土壤后存活，表明它们得到了有效的炼苗。采用随机扩增多态性DNA（RAPD）和小卫星区域定向扩增（DAMD）等DNA分析（基于PCR的标记）来评估离体培养的植株，这些植株产生了单态条带模式，证实了遗传稳定性。此外，未检测到再生植株与供体植株之间的倍性流式细胞仪图谱变化。这种珍贵药用植物的离体再生将为制药生物技术开辟新途径，通过提供一个非常规的稳定系统进行大规模繁殖，并可能有效地用于基因操作以提高生物活性成分。
