# Halophyte _Artemisia caerulescens_ L.: Metabolites from In Vitro Shoots and Wild Plants
## 2 Results and Discussion  
### In Vitro Shoot Proliferation  
_A. caerulescens_ grows spontaneously in the San Rossore Estate, in salty soil close to the sea. Few studies are available on its ecological adaptability or on its growth strategy [8; 12]. Aerial parts were harvested in June and September 2021, at the vegetative and flowering stages, and then used for in vitro shoot proliferation to avoid the depletion of the wild plants for future analysis. The same \"mother\" plants were also used for the biochemical and phytochemical analyses. The sterilization step was repeated for each single mother plant; the obtained results are summarized in Table 1. The initial contamination ranged between 10% and 90% of the explants and appeared a few weeks after the sterilization process. The high contamination could be due to the natural and protected habitat of the spontaneous plants and the lack of a pre-treatment with pesticides before the in vitro cultivation, as reported in some literature [18]. The shoots proliferated in either Murashige Skoog basal medium [19] (MS0) or in MS with the addition of cytokinin (MS-BA). Shoot induction was performed with the addition of cytokinin 6-benzyl-aminopurine (BA) as a unique growth regulator, which was a pivotal experiment investigating the ability of A. _caerulescens_ in shoot proliferation. After 4 weeks of cultivation, the MS0 explants showed a lower proliferation (one shoot/explant), while the explants grown in the presence of BA showed better proliferation, with 3-5 shoots in independent experiments. However, the addition of BA produced shorter shoots (1.2-1.5 cm in length) than those of MS0 (2.67-3.5 cm) (Figure 1c,d, respectively). Therefore, other shoots were cultured with MS, combined with different concentrations of BA (1, 2, or 4 m). The highest proliferation of shoots was obtained with the lowest BA concentration (8.1 shoots per explant), followed by the concentration of 2 m and 4 m BA. The highest shoot length was achieved with the use of the MS0 medium (range, 2.67-3.5 cm), and the shortest shoots (0.76 cm) with the 4 m BA. The effect of the addition of cytokinin is well-known to increase the number of shoots in other species. In _A. vulgaris_, the shoots proliferated in liquid MS with 0.44-8.88 m BA, and the best effect on shoot multiplication was obtained with the addition of 4.44 m BA, producing 85.5 shoots/explant at 500 mL flask capacity and with an average length of 12.2 cm [20]. The extraordinary data differed from our results due to the methodology and the species used. Usually, the solid medium \begin{table}
\begin{tabular}{l c c c c c} \hline \hline \multicolumn{1}{c}{\multirow{2}{*}{**Mother Plant**}} & **Number of** & **Contaminated** & **Developed Shoots** & **Number of** & **Shoots Length** \\  & **Explants** & **Explants (\%)** & **(\%)** & **Shoots/Explant** & **(cm)** \\ \hline M1 (MS0) & 18 & 32 & 88.9 & 1 & 2.67 \(\pm\) 1.7 \\ M2 (MS0) & 10 & 0 & 100 & 1 & 3.5 \(\pm\)1.6 \\ M3 (MS0-BA) & 20 & 90 & 0 & 0 & 0 \\ M4(MS-BA 2 m) & 10 & 10 & 3 & 3 & 1.2 \(\pm\) 0.4 \\ M5(MS-BA 2 m) & 10 & 10 & 5 & 5 & 1.5 \(\pm\) 0.5 \\ \hline MS0 & 10 & 0 & 100 & 2.1 \(\pm\) 1.28 C & 3.18 \(\pm\) 0.26 A \\ MS-BA 1 m & 10 & 0 & 100 & 8.1 \(\pm\) 1.63 A & 1.12 \(\pm\) 0.14 C \\ MS-BA 2 m & 10 & 0 & 100 & 5.4 \(\pm\) 1.08 B & 1.5 \(\pm\) 0.12 B \\ MS-BA 4 m & 10 & 0 & 100 & 4.6 \(\pm\) 1.27 B & 0.76 \(\pm\) 0.11 D \\ \hline \hline \end{tabular}  
* The superscript, uppercase letters (A–D) indicate statistically significant differences between the samples. The statistical significance of the relative abundances was determined by the [PERSON]’s post hoc test, with \(p\leq 0.05\).  
\end{table} 
Table 1: In vitro culture of _A. caerulescens_. Single mother plants (M) were used as initial explants, and their contamination was observed (%). Effect of different culture media on shoot development (%). Proliferation and length of shoots (cm) after 4 weeks of cultivation. Data are presented as means \(\pm\) SD (\(n=10\)). is used for shoot induction and proliferation. In _A.