In vitro culture has been recognized as a potential plant clonal propagation tool for a broad variety of commercial, ornamental, and medicinal species, with applications in both industrial and academic laboratories. In this study, we describe somatic embryogenesis and plant regeneration protocol for “peperina” plants (Minthostachys verticillata (Griseb.) Epling). In vitro shoots developed via shoot apex extracted from greenhouse-grown plants were cultured on shoot elongation medium (ShM) consisting of Murashige and Skoog (MS) basal salts supplemented with myoinositol, thiamine, and benzyladenine. Shoot apexes were disinfected with 70% ethanol for 5 min and 0.26 sodium hypochlorite (w/v) for 5 min, before the initiation of in vitro culture. For somatic embryo (SE) induction, leaves collected from 2-mo-old in vitro raised shoots were cultured on MS basal medium supplemented with benzyladenine and two different concentrations of coconut water (CW) until SE developed. Using 2.5% CW-supplemented medium, 100% of cultured leaves developed SE and 89.3% of the leaf explants developed plantlets. The resulting embryos germinated on the same medium, and the plantlets obtained were transferred onto ShM until they developed 3 to 4 leaves. To increase the low root development, shoots were transferred into fully hydrated perlite for rooting and then transplanted into soil-perlite containing pots for hardening. This protocol provides the first step to supply the demand and simultaneously protect the natural populations of Minthostachys verticillata from overexploitation. Furthermore, this protocol provides the first step for crop improvement by genetic modification (editing or transgenesis).

体外培养已被认为是用于各种商业，观赏和药用物种的潜在植物克隆繁殖工具，并在工业和学术实验室中得到应用。在这项研究中，我们描述了“ peperina”植物（Minthostachys verticillata（Griseb。）Epling）的体细胞胚发生和植物再生方案。通过从温室植物中提取的茎尖发育的体外芽，在由Murashige和Skoog（MS）基础盐以及肌醇，硫胺素和苄腺嘌呤组成的茎伸长培养基（ShM）中培养。芽尖用70％乙醇和0.26次氯酸钠（w / v）消毒5分钟）5分钟，然后开始体外培养。对于体细胞胚（SE）诱导，从2个月大的体外收集叶片在补充了苄基腺嘌呤和两种不同浓度的椰子水（CW）的MS基础培养基上培养高芽，直至形成SE。使用2.5％CW补充的培养基，100％的培养叶发育出SE，89.3％的叶外植体培养出了小植株。所得的胚在相同的培养基上萌发，将获得的小植株转移到ShM上，直到发育3至4片叶子。为了增加低根发育，将芽转移到完全水合的珍珠岩中生根，然后移植到含有土壤珍珠岩的盆中进行硬化。该协议为满足需求并同时保护Minthostachys verticillata的自然种群提供了第一步来自过度开发。此外，该协议为通过遗传修饰（编辑或转基因）改良作物提供了第一步。