Successful in vitro micropropagation depends on several crucial factors: donor plant condition, explant selection, sterilization methods, and growth medium composition. The MS medium, rich in nutrients and organic compounds, has proven effective for many plant species (Khatri et al., 2019). Proper sucrose levels and growth regulator selection are vital for culture development. Notably, even explants from very old plants can exhibit totipotency and be successfully propagated in vitro, demonstrating that age is not always a limiting factor in micropropagation (Szyp-Borowska et al., 2020).
This study established the first reliable in vitro propagation method for L. wightianum using nodal explants. The protocol achieved an 80% explant survival rate on MS media supplemented with growth regulators. Adventitious bud proliferation was successful using BAP (2 mg/L) and Kinetin (1 mg/L). This high cytokinin concentration promoted multiple adventitious shoot formation, consistent with findings in other species like Robinia pseudoacacia (Szyp-Borowska et al., 2020). The effectiveness of cytokinins in promoting axillary bud growth and reducing apical dominance in broad-leafed plant cultures was noted. Similar successful micropropagation using MS medium has been reported for other woody species, including Cotoneaster sp (Monier and Ochatt, 1995), Betula lenta (Rathwell at al., 2016), and Citronella mucronata (Guerra et al., 2022).
Rhizogenesis, a complex process of root development in tissue culture, is heavily dependent on growth regulators, particularly auxins (IAA, IBA, and NAA). These auxins promote root initiation by stimulating meristem formation in cells (García-Garrido et al. 2000). Selecting the appropriate auxins and their concentrations is crucial for successful rooting. At wound sites, auxins cause cell dedifferentiation, leading to new root meristem formation. Auxins play diverse roles in rooting processes, including increasing root numbers and forming adventitious roots (Guan et al., 2019). The effectiveness of different auxin types can vary based on their interactions with auxin receptors, which may be influenced by cultivar (Olatunji et al., 2017). Additionally, auxins can speed up rooting and improve root quality (Zayova et al., 2014). In some woody plants like Robinia pseudoacacia, NAA has been found to stimulate root production more effectively than IBA (Kovačević et al. 2014).
In the current study, in-vitro rooting of L. wightianum exhibited recalcitrance, achieving only a 40% rooting rate with low concentrations of the auxin IBA. In contrast, higher rooting percentages have been reported for other commercially important plants. For instance, Singh and Agarwal (2016) recorded high rooting percentages for Simmondsia chinensis, while an experiment by Delgadillo-Díaz et al. (2013) on Quercus species reported rooting rates of 83.3–100%. Moreover, several studies have documented good rooting percentages using low concentrations of IBA. Guerra et al. (2023) achieved 85% rooting in Peumus boldus Mol with 9.84 µM IBA, Guo et al. (2019) achieved 84–100% rooting in Vaccinium corymbosum with 2.46 µM to 4.92 µM IBA. In addition to extended in-vitro culture with low auxin concentrations, transient dipping into high concentrations of auxins, particularly IBA, remains a viable method for achieving prompt rooting in woody trees.
In our study, approximately 55% of snap-dipped shoots survived under soil conditions. Bhargava et al. (2018) conducted similar experiments on sandalwood (Santalum album), achieving 50% rooting. Additionally, the same authors reported a 1.8-fold increase in rooting rates for chestnut propagation when shoots were transiently immersed in 9 g/L IBA for 10 seconds, and for olive (Olea europaea ‘Manzanilla’) treated with 3 g/L IBA (Khajehpour et al. 2014). Pulse treatment with IBA has also been successfully applied in the adventitious rooting of Maytenus emarginata (Rathore et al. 1992) and Tectona grandis (Siril & Tewari 1999). Furthermore, in-vitro rooting with low IBA concentrations resulted in a greater number of roots per plant, which were also more robust in size. Previous studies indicate that plantlets cultured in media with exogenous adjuvants are healthier compared to those subjected to transient immersion (Vielba et al., 2019).
However, sugar and salt concentration (low or high) in the media can severely affect the basic root development process. In our experiment, the roots grew spontaneously in the half and quarter strength growth medium with a reduced concentration of sucrose (2%, instead of 3%). Researchers found that reducing the concentration of sucrose to 2% (instead of 3%) was beneficial in achieving callus-free rooting (Dhabai et al. 2010). Being an energy demanding process, an exogenous supply of carbohydrate is essential for root formation and transformation of plant from heterotrophic mode of nutrition to autotrophic mode is also an important issue (Serret et al. 1997).
In current research, it was found that using a ¼ strength MS medium, which has reduced nutrients, led to better root growth compared to using a ½ strength MS medium, both in both ex vitro and in vitro rooting trials. Previous studies have shown that the reduced nutrient content in the ¼ strength MS medium promotes root growth by activating local and systemic signaling pathways. This medium also results in less growth of the aerial portion of the plant, but encourages more branching and development of the root system, thus enhancing mineral nutrient absorption (Manokari 2021). Since full strength or ½ strength MS medium creates a high osmotic environment, transitioning plantlets directly from this medium to soil can cause them to collapse or die. To address this, researchers have been reducing the strength of the MS medium to ½ or even ¼ strength to lower the osmotic pressure. Furthermore, for proper root initiation and growth, cells need to effectively uptake water, which can be hindered by the high osmotic pressure of full strength or ½ strength MS medium. Therefore, MS medium with lower osmotic pressure remains the preferred choice for rooting experiments (Dönmez et al. 2022).
Adaptation of juvenile plantlets to an alternative substrate significantly enhances their survival capacity in a new environment. In our study, we placed the juvenile plantlets in polybags containing sand and FYM (Farm Yard Manure) for 15–20 days to facilitate their primary hardening and adaptability to soil pots. This approach ensured high humidity, aiding in their acclimatization. After this period, the plants were subjected to periodic pulse treatments under full sunlight for 2–3 days, resulting in a survival rate of about 88 to 93% within two months. Comparable results were reported by Tzatzani et al. (2023), who observed an 83% survival rate for Psidium guajava during the acclimation phase using a substrate mixture of equal parts vermiculite, peat, and perlite. In contrast, Kaviani et al. (2023) reported a 90% survival rate for Pyrus communis during the acclimation phase with a substrate mixture of peat and perlite. Similarly, Kaviani et al. (2023) demonstrated a 92% survival rate for Pyracantha angustifolia using a perlite and peat mixture in a 2:1 ratio. Remarkably, Citronella mucronata, a woody species, achieved a 100% survival rate on a peat and perlite mixture after 30 days of careful acclimatization practices in a greenhouse environment.