The observations of the study highlighted the importance of growth regulators and induction period for the establishment of somatic embryogenesis in P. santalinus. The importance of auxins, especially 2,4-D, was already confirmed for somatic embryogenesis in case of woody (Mehta et al. 2011; Nunes et al. 2018) and Fabaceous trees (Han and Park 1999; Trigiano et al. 1999; Buendı´a-Gonza´lez et al. 2012). In P. marsupium, Husain et al. (2010) used hypocotyl explant and showed the formation of somatic embryos by 5 µM 2,4-D with 1 µM BAP. Lakshmi Sita et al. (1980) obtained embryogenic callus upon induction with 1 mg/l 2,4-D using shoot pieces as explant in sandalwood. Our observation also follows these results proving the efficiency of 2,4-D for somatic embryogenesis. Many researchers assessed the potency of other auxins for somatic embryogenesis in woody species (Dunstan et al. 1995; Kendurkar et al. 1995) and Fabaceae plants (Canhoto et al. 2006). Auxins proved to be an efficient group of PGRs for somatic embryogenesis, probably because of its role in cell cycle, division, and differentiation (Lemes da Silva et al. 2021). 2,4-D reported as one of the most suitable auxins for somatic embryogenesis might be because of its role in DNA hypermethylation (Ebrahimi et al. 2018). The result of the present study reported better somatic embryogenesis when cytokinin and auxins were used in combinations, whereas, cytokinin alone found to be inefficient for the induction. The similar result has also been reported by Canhoto et al. (2006) in carob, where they observed somatic embryogenesis when BAP and IAA have been used in combinations, whereas, the individual use of hormones proved ineffective. Auxins and cytokinin both play a vital role to regulate the embryogenic response which also depends upon the internal hormonal supply that varies from species to species and explant to explant (Verma et al. 2016). In the current study cotyledon explants from germinated immature zygotic embryo were used for the production of somatic embryos. Immature cotyledons provide a developmental window that can be utilized for somatic embryogenesis (Trigiano et al. 1999). Canhoto et al. (2006) showed the importance of explant for induction of somatic embryogenesis while working with carob. The difference of in-vitro condition or the genotype of the plant and the explant have a significant effect on the successful induction and maintainance of somatic embryogenesis (Canhoto et al. 2006). Gulzar et al. (2020) reported that though with the advancement of technology the difficulty in establishment of somatic embryogenesis has been overcome, but still, most of the woody species are either remain recalcitrant or respond poorly for the same. They also mentioned the incapability of embryonic cells to develop into complete plantlets. The same has been validated by our results with lesser number of the globular-staged embryos, cotyledonary-staged embryos and plantlets.
The period for induction of somatic embryogenesis varies in different plant species. Some plants require a short period of induction (Sharry et al. 2006), whereas others require prolonged induction (Wang et al. 2003). In the current study the somatic embryos were obtained after prolonged induction of 12 weeks. Singh and Chand (2003) obtained 26.5 average number of somatic embryos after 15 weeks of culture in half-strength MS media. Sucrose is also an important factor for the induction of somatic embryogenesis because it acts as an osmotic stress (Verma et al. 2016). The author’s group also worked with 3% sucrose on the same explant and resulted in non-embryonic callus, the results are already being published (Chakraborty et al. 2022). Our current observations and results are in accordance with Verma et al. (2016), who obtained a 10% increase in embryogenic callus frequency with 5% sucrose. Higher sucrose concentration was also reported in other plant species, e.g., grapevine (Li et al. 2014), Hevea brasiliensis (Srichuay et al. 2014), sandalwood (Herawan et al. 2014), Amorphophallus konjac (Li et al. 2021) and Cambod tea (Mishra et al. 2022). Sucrose interacts with PGRs and help in growth and development process of plants. It also helps in metabolic processes of plants (León and Sheen 2003; Skylar et al. 2011). Koch (1996) reported the function of sucrose in transcriptional, post-transcriptional, and post-translational processes. Sucrose enhances the expression of the genes controlled by promoters like patatin and phloem-specific rolC (Jeferson et al. 1990; Yokoyama et al. 1994).
The success of micropropagation through somatic embryo formation depends on its development and final conversion to plantlets, steps which remains a challenging work for many woody species (Isah 2019). The same has been noticed for P. santalinus which prevented from getting a large number of plantlets from the conversion of somatic embryos. Some somatic embryos evolved in shoots or roots on the conversion medium, indicating incomplete maturation and incapability in completing the morphogenic process. Weaver and Trigiano (1991) worked with a Fabaceae plant, Cladrastis lutea, and established the lack of somatic embryo conversion to plantlet. The conversion problem might be due to the anomalous shoot apex formation, which in turn depends on the type of auxin and the duration of PGR it is exposed to (Weaver and Trigiano 1991). Similar observations have also been reported by Canhoto et al. (2006) in carob, a Fabaceae species stating the defect in embryo maturation and meristem formation. The mature cotyledonary stage somatic embryos germinated to complete plantlets with prominent roots and shoot growth in the basal medium supplemented with 3% sucrose. Singh and Chand (2003) worked with a timber-generating leguminous plant, Dalbergia sissoo Roxb., and successfully achieved 50% of embryo conversion using cotyledon as explant. They used 10% sucrose for the maturation and 2% sucrose for the germination of the matured somatic embryos. High concentration of sucrose prior to the germination improves the process as it acts as a signal for biogenesis of stored proteins (Singh and Chand 2003). On the contrary, the high sucrose concentration may inhibit germination, if present in the germination medium, due to osmotic shock (Verma et al. 2016). As observed by our experiment that the matured somatic embryos generated from the combinations of PGR treatments converted into plantlets showed the importance of induction medium on successful conversion of somatic embryos. Lemes da Silva et al. (2021) reported that the treatment of 18.1 µM 2,4-D + 4.5 µM BAP resulted in maximum somatic embryos and also being the only treatment that regenerated plantlets after their transfer to basal media. Hence, the development of somatic embryos and its successful conversion also depends on various factors like the explant, growth regulators, and duration of the use of growth regulators.
The histological analysis helped to differentiate between embryogenic and non-embryogenic callus formed simultaneously from the same explant. It showed the presence of a dense cytoplasm within the parent cell wall, a characteristic feature of somatic embryos. The same characteristics were also evident in embryogenic cells of other species (Nunes et al. 2018; Sun et al. 2021). The polar migration of embryonic callus towards the tip consisting of meristematic cells was also evident during somatic embryogenesis in zygotic embryo of Cunninghamia lanceolata (Hu et al. 2017). As acetocarmine is used to detect DNA and chromatin, it can easily differentiate embryonic cells from non-embryonic cells and also from the attached suspensor. It helps to tract the polarly movement of the embryonic callus towards the tip (Hasbullah et al. 2007). The attached suspensor with embryonic cells, a characteristic feature of somatic embryos, has also been shown by Xia et al. (2021) during the work on masson pine (Pinus massoniana). An early phase of somatic embryo development with dense cytoplasm at embryonal end with long suspensor has been shown by Arya et al. (2000) while working with Pinus roxburghii Sarg. Many other researchers also used acetocarmine to differentiate between proembryonal, embryonal, and non-embryonic tissue (Fráterová et al. 2013; Hazubska-Przybył et al. 2020).