The conversion percentage, number of leaves and shoots, stem diameter, plant height, shoot and root dry mass of SS of A. oleracea differed between treatments (Table 1). The higher percentage of SS conversion with all BAP doses than in the control, is due to the stimulation of cell division and early vascularization of tissues by this growth regulator, facilitating the translocation of nutrients from the culture medium to the plant (Sosnowski et al. 2023). The technique with growth regulators and non-embryogenic explant (vegetative segments) has also been reported for Althaea officinalis, Plumbago rosea and Tylophora indica (Gantait et al. 2017; Naz et al. 2018; Prakash et al. 2018). The high efficiency of this method in producing SS is also due to the high cellular competence at early stages of organ development and tissue differentiation (Bustillo-Avendaño et al. 2018; Priyadharshini et al. 2020; Bertsouklis and Tsopela 2023).
Table 1
Summary of the analysis of variance of the data of biometric characteristics evaluated in the development of synthetic seeds of Amella oleracea in the different treatments
| | ----------------------------------Mean Square--------------------------------- | |
FV | gl | CO | NL | S | SD | H | SDM | RDM | |
Trat. | 4 | 0.056* | 163.50** | 1.39** | 8.90** | 50.50** | 5.03** | 9.7×10− 5** | |
Res. | 50 | 0.105 | 18.41 | 0.1726 | 0.326 | 3.56 | 3.44×10− 5 | 8.73×10− 6 | |
Mean | - | 0.84 | 13.91 | 1.50 | 1.96 | 7.22 | 0.028 | 0.0063 | |
CV (%) | - | 37.36 | 30.85 | 27.66 | 29.10 | 26.12 | 20.68 | 46.96 | |
** and * significant effects at 0.1% (p < 0.001) and 1% (p < 0.01) probability levels, respectively, by the F test. CO Conversion percentage, NL number of leaves and S shoots, SD stem diameter, H plant height, SDM shoot and RDM root dry, Treat treatments, Res residues.
The percentage of SS conversion (CO) in A. oleracea plantlets was higher in the treatments with the BAP growth regulator, 81.8–100.00%, than in the control (Table 2).
Table 2
Biometric characteristics of Acmella oleracea synthetic seeds produced with various concentration of 6-benzylaminopurine
BAP (mg l− 1) | 0.000 | 0.025 | 0.050 | 0.100 | 0.200 | DMS |
CO (%) | 45.452.22b | 81.840.45a | 100.00.01a | 90.930.15a | 100.00.01a | 39.18 |
NL | 7.556.72b | 14.182.14a | 14.641.86a | 18.003.10a | 15.185.42a | 5.1774 |
SN | 0.930.67b | 1.550.17a | 1.640.24a | 1.900.26a | 1.490.51a | 0.5012 |
SD (mm) | 2.861.44a | 1.710.20bc | 1.430.09c | 1.670.11bc | 2.140.51b | 0.6889 |
H (cm) | 3.412.83b | 8.131.12a | 8.261.16a | 8.450.73a | 7.872.58a | 2.2763 |
SDM (g) | 0.0220.006c | 0.0270.004bc | 0.0270.005abc | 0.0340.007a | 0.0320.007ab | 0.0071 |
RDM (g) | 0.0010.001b | 0.0070.002a | 0.0060.002a | 0.0080.003a | 0.0080.005a | 0.0036 |
Means, per line, followed by the same letter do not differ by Tukey's test. Superscript values represent the standard error. CO- Conversion percentage, NL- number of leaves and S- shoots, SD- stem diameter, H- plant height, SDM- shoot and RDM- root dry, Treat- treatments, Res- residues.
The number of leaves and shoots, plant height and root dry mass were higher for plantlets originated from SS in the BAP treatments than in the control (Table 2). Shoot dry mass was 54.5% higher for plantlets originated from SS in the treatment with 0.100 mg l− 1 of BAP than in the control. The highest number of leaves and shoots, plant height and shoots and roots dry mass of A. oleracea plantlets using BAP agrees with that observed in vitro culture of Artemisia laciniata (Astereceae) with 0.1 mg l− 1 of this growth regulator and is due to the greater organogenesis in A. oleracea plantlets (Kodym et al. 2018). BAP acts in the regulation of plant growth, cell division, regeneration, organ formation and induction of multiplication and development of shoots in SS, as reported for the in vitro cultivation of Achillea fragrantissima and Ansellia africana with concentrations of 0.1 and 0.225 mg l− 1 of BAP, respectively (Bhattacharyya et al. 2018; Goda et al. 2023; Asghar et al. 2022). Synthetic growth regulators of the cytokinin class, including BAP, increase the translocation of nutrients from the culture medium, photosynthesis and the production and transport of photoassimilates increasing shoot and root development (Sosnowski et al. 2023)
The stem diameter of A. oleracea was greater in the control than in the treatments with different BAP concentrations (Table 2). The larger stem diameter of the A. oleracea seedlings in the control than in the treatments with BAP is due to the smaller number of shoots, reducing the mobilization of nutrients from the culture medium and photoassimilates for different parts of the plant, compared to the treatments with BAP. The greater number of shoots increases the competition for photoassimilates between expanding young leaves, decreasing mass accumulation per shoot and reducing plantlets diameter, as reported with nodal segments of Manihot esculenta (Alves et al. 2018; Feyisa 2023).
The stomata of A. oleracea plantlets are anomicytic (subsidiary cells absent), ellipsoid with an average length of 40 µm and were open and well defined (Fig. 3A, B, C, D and E). Epicuticular wax was not observed in the abaxial epidermis of A. oleracea leaves (Fig. 3F, G, H, I and J). The presence of anomicytic-type stomata, with an ellipsoid shape, open and well defined, is associated with their normal functionality in in vitro culture (Khan et al. 2002; Afreen 2005). Environmental conditions inside the test tube did not affect stomatal size, and high humidity kept them open. The size and shape of the stomata in the control and treatments with BAP indicate that this growth regulator did not alter them in A. oleracea plantlets produced in vitro, as already reported in its ex vitro cultivation (Matyushin and Evdokimova 2017). The absence of epicuticular wax on the abaxial surface of A. oleracea leaves is due to morphological adaptations, associated with the in vitro culture condition, reducing transpiration control mechanisms and facilitating the continuous loss of water from the leaf (Lando et al. 2016; Vieira et al. 2020; Garvita and Wawanngningrum 2020).