Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
Appl Biochem Biotechnol. 2013 Apr;169(7):2004-15. doi: 10.1007/s12010-013-0104-7. Epub 2013 Jan 26.
A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg L(-1) 6-benzyladenine (BA) along with 0.5 mg L(-1) gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg L(-1) BA in combination with 0.25 mg L(-1) α-naphthalene acetic acid (NAA) and 0.25 mg L(-1) 2,4-dichlorophenoxyacetic acid or 0.5 mg L(-1) indole butyric acid (IBA) along with 0.25 mg L(-1) NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg L(-1) thiodiazoran or 1.5 mg L(-1) IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle "piperine." The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.
已建立了一种可靠的体外再生系统，用于经济且具有重要药用价值的胡椒（Piper nigrum L.）。在添加不同浓度植物生长调节剂的 Murashige 和 Skoog (MS) 培养基上，从叶片部分促进愈伤组织和芽的再生。在培养 4 周后，在含有 1.0 mg L(-1)6-苄基腺嘌呤（BA）和 0.5 mg L(-1)赤霉素的 MS 培养基中孵育的外植体中观察到更高的愈伤组织产生（90%）。此外，还记录到在 1.0 mg L(-1)BA 与 0.25 mg L(-1)α-萘乙酸（NAA）和 0.25 mg L(-1)2,4-二氯苯氧乙酸或 0.5 mg L(-1)吲哚丁酸（IBA）与 0.25 mg L(-1)NAA 和吲哚乙酸的组合下，85%的愈伤组织也具有生芽反应。继 4 周后，将愈伤组织继代培养到补充有 1.5 mg L(-1)硫代二唑烷或 1.5 mg L(-1)IBA 的 MS 培养基上，诱导 100%的芽反应。在含有不同浓度植物生长素的培养基上获得生根的幼苗。抗氧化酶活性[超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）和抗坏血酸过氧化物酶（APX）]表明，再生植物中的 SOD 明显高于其他组织。然而，在愈伤组织中 POD、CAT 和 APX 高于其他组织。建立了一种高效液相色谱（HPLC）指纹分析方案，用于不同胡椒（Piper nigrum L.）体外再生组织的质量控制。在分析过程中，大多数常见峰代表活性成分“胡椒碱”。化学含量，特别是胡椒碱，从愈伤组织培养到整个幼苗再生都有变化。基于色谱峰的偏差，体外再生的幼苗表现出与驯化幼苗几乎相似的胡椒碱谱。这里建立的体外再生系统和 HPLC 指纹分析为体外幼苗的质量控制带来了新的方法，为生物多样性保护提供了具有重要应用前景的代谢产物。
