Somatic embryogenesis and plantlet regeneration in red sandalwood (Pterocarpus santalinus)

Cotyledonary segments from the germinated immature zygotic embryos were used for the somatic embryogenesis of red sandalwood (Pterocarpus santalinus). The explants were established on Murashige and Skoog (MS) medium containing 5% sucrose and the combination of 6-benzylaminopurine (BAP), 2,4-Dichlorophenoxyacetic acid (2,4-D), and α-Naphthaleneacetic acid. All the treatments responded to callus induction with a 36–97% frequency range. The maximum embryogenic frequency (69.44%) was obtained when 0.1 mg/l BAP + 2 mg/l 2,4-D and 0.1 mg/l BAP + 4 mg/l 2,4-D combinations were used. When the explants were treated with individual growth regulators, the maximum embryogenic frequency (58.33%) was produced by 4 mg/l 2,4-D. BAP was completely ineffective for somatic embryogenesis when used individually. The average number of globular-staged somatic embryos ranged between 1 and 5 (irrespective of the treatments). The maximum number of the cotyledonary-staged somatic embryos (2.85) was obtained with the treatment 0.1 mg/l BAP and 2 mg/l 2,4-D. The maximum plantlets were developed (1.30) when the cotyledonary-staged embryos from 0.1 mg/l BAP and 2 mg/l 2,4-D were transferred to MS basal medium. The plantlets obtained were acclimatized and showed 100% survival in the greenhouse condition. The embryonic cells have been histologically distinguished from non-embryonic cells with dense cytoplasm and a long suspensor. The induction, maturation, and germination of somatic embryos were challenging, suggesting the need for molecular approaches through proteomic expression for mass production and understanding of the evolution, structure, and genetic organization of the plant species. 2,4-D and NAA, individually or in combination with BAP, has a significant effect on the embryonic potential of cotyledon explant of P. santalinus.


Introduction
Pterocarpus santalinus is a woody tree of the Fabaceae family, endemic to the Deccan part of the Indian peninsula, and it is cultivated in China, Pakistan, Sri Lanka, and Philippines (Ahmedullah et al. 2019). It is an endangered species with exceptional timber quality, listed in Endangered A2cd ver 3.1 of the IUCN Red List. The plant has been smuggled Ahmedullah 2021). The hardwood is 16% saturated with a red-colored dye known as santalin, which is used as a coloring agent in the pharmaceutical, food, and leather industries (Arunkumar and Joshi 2014). The species is also a rich source of numerous phytometabolites, including glycosides, flavonoids, alkaloids, tannins, phenols, saponins, sterols, and triterpenoids (Arunakumara et al. 2011;Navada and Vittal 2014;Bulle et al. 2016). Shree et al. (2019) have reported the presence of two antitumor molecules, savinin and calocedrin. The plant products are in use as a folk medicine to cure skin infections, blood diseases, vision problems, diabetes, bile problem, and insect bites (Arunakumara et al. 2011;Azamthulla et al. 2015;Keshavamurthy et al. 2018;Rao et al. 2019;Shree et al. 2019). The extracts of red sandalwood have the potential to treat diabetic neuropathy (Halim and Misra 2011) and also help in angiogenesis (Jadhav et al. 2011) and vasculogenesis (Jadhav et al. 2012). Because of its curative potential, the plant has been considered for the 21st meeting of the plant committee by RST (Review of Significant Trade) as a priority species for review (UNEP-WCMC 2017). The natural regeneration of the plant is difficult due to hard pod, low fruit formation, stringent habitat, lengthened dormancy, and impoverished seed development (Rao and Raju 2002;Hegde et al. 2012;Arunkumar and Joshi 2014). Conventional regeneration methods like grafting, root cutting, and air layering proved futile (Chaturani et al. 2006;Vijayalakshmi and Renganayaki 2017;Renganayaki et al. 2020). In this context, in-vitro techniques offer an alternative to red sandalwood propagation. Micropropagation of P. santalinus through direct organogenesis has been reported by many researchers (Anuradha and Pullaiah 1999;Arockiasamy et al. 2000;Prakash et al. 2006;Rajeswari and Paliwal 2008;Padmalatha and Prasad 2008;Balaraju et al. 2011;Warakagoda and Subasinghe 2013;Chen et al. 2019). In comparison, the regeneration of the plant species via callus formation was reported by Chakraborty et al. (2022). The difficulty of obtaining somatic embryos in woody species is already an established fact (Isah 2019;Gulzar et al. 2020). Despite the problems reported, somatic embryogenesis has been reported in many woody species (Hu et al. 2017;Nunes et al. 2018;Sun et al. 2021;Xia et al. 2021;Yan et al. 2021). Individually, the process has also been achieved in many leguminous plants (Singh and Chand 2003;Buendía-González et al. 2012;Viji et al. 2012;Kaul et al. 2014;Nolan et al. 2014), including P. marsupium (Husain et al. 2010). As P. santalinus is an endangered and commercially valuable species, somatic embryo formation is needed for its germplasm conservation. Somatic embryogenesis also helps in clonal propagation because somatic embryos have a single-cell origin, an essential characteristic that will help hold the mother plant's superior traits (Guan et al. 2016). To our knowledge, there is no report of somatic embryogenesis in P. santalinus. This study documents plantlet regeneration by establishing somatic embryogenesis in red sandalwood and histological analysis to differentiate embryonic and nonembryonic cells from cotyledon explants.

Plant material and surface sterilization
Immature green-winged pods of P. santalinus were collected after 45 days of anthesis from a 15-year-old plant growing in the Andhra Pradesh Red Sandalwood Forest Patch (Latitude N 17° 53′ 9.955″, Longitude E 83° 19′ 42.648″) (Fig. 1A). The wings were excised from the pods (Fig. 1B) and rinsed under tap water for 10-15 min, followed by disinfection with 1% (v/v) Savlon and 20 drops of Tween 20 for 10 min. After washing under running tap water, the surfaces were sterilized with 70% ethanol for 2 min, followed by the treatment with 0.1% HgCl 2 for 15 min inside Laminar Air Flow (LAF; KEMI, India). Finally, the pods were rinsed 4-5 times for 30 s to 1 min with sterilized double distilled water. The immature zygotic embryos were dissected by removing the hard outer layer of the pods with the help of a wire cutter (Fig. 1C) and placed on MS (Murashige and Skoog 1962) medium for germination. After 28 days, the 0.5-1 cm cotyledon sections were sliced out from the 5-6 cm height plantlet and used as an explant to induce somatic embryos (Fig. 1D).
Cotyledonary-staged somatic embryos were transferred to the full-strength MS basal media and the media containing BAP (0.5, 1.0, and 2.0 mg/l) and 2,4-D (0.5, 1.0, and 2.0 mg/l) individually and in combinations. All the treatments were added with 3% (w/v) sucrose and 1% (w/v) agar powder for germination and kept in an air-conditioned tissue culture room maintained at 25 ± 2 ℃, 16/8 h (light/ dark) photoperiod receiving 70 μM/(m 2 s) with 40 W cool white fluorescent tube lights for 3-4 weeks.

Acclimatization
The plantlets obtained from converting somatic embryos from all the applied treatments were further acclimatized in the laboratory and greenhouse conditions. First, the plantlets were carefully drawn out from the agar media not to break their root or root hairs. The roots of the plantlets were then cleaned lightly under tap water to remove any attached media to it. After that, the roots were treated with an antifungal, Bavistine (0.5%, w/v), for approximately 3 min. After the treatment, the plantlets were shifted to a transparent plastic glass containing a combination of garden soil and peat (1:1, w/w). The humidity was maintained by covering the plantlets with a plastic glass in an upside-down position before shifting to the culture room (25 ± 2 ℃ and 16/8 h (light/dark) photoperiod) for 1-2 weeks. During this period, the humidity was progressively decreased by removing the upper glass cover. The well-acclimatized plantlets were selected for further acclimatization, wherein the plantlets were shifted to the laboratory conditions for another 4 weeks. The soil mixtures used during this period were garden soil and peat in a 1:1 (w/w) ratio. As the plants grew in height, the acclimatized plantlets were shifted to bigger pots progressively and finally transferred to the greenhouse for further development.

Growth measurement
The growth for callus and somatic embryo development were measured based on the callogenic and embryogenic frequency, respectively. The embryogenic responses were further measured as (i) the average number of globularstaged embryos, (ii) the average number of cotyledonarystaged embryos, and (iii) the average number of plantlets per treatment. The response parameters were calculated as follows:

Histological studies
The embryonic cells were observed fresh by placing them on a glass slide with the help of an inoculating loop. The cells were then stained with 2% (w/v) acetocarmine for 30 s. The staining solution was washed with distilled water, and 1-2 drops of glycerol were added before mounting with a cover slip. The microscopy was performed under light microscopy (CH20 i, Olympus, Japan).

Statistical analysis
The results for all the experiments were statistically analyzed using SPSS (IBM, version 19.0). The mean with the standard error was calculated with Microsoft Excel (2016 version) and presented in a tabular form. The average callogenic frequency, embryogenic frequency, globular-staged embryos per treatment, cotyledonary-staged embryos per treatment, and plantlets generated per treatment were subjected to analysis of variance (ANOVA). Significant differences among Callogenic frequency (%) = ( No. of explants that produced callus/Total number of explants ) × 100 Embryogenic frequency (%) = ( No. of explants that produce embryogenic callus/Total number of explants ) × 100 means were evaluated at p ≤ 0.05 level by a Duncan multiple comparison test.

Somatic embryogenesis induction
The experiments were divided into individual treatments or combinations of auxins and cytokinin (BAP, 2,4-D, and NAA). It was observed that though all the concentrations of BAP (when used alone) were significantly effective for callus formation, they were unresponsive for somatic embryo formation. The maximum callogenic frequency (95.37 ± 2.44%) was obtained at 0.1 mg/l BAP (Fig. 2). The individual treatment with auxins, either 2,4-D or NAA, was found effective (at significance p < 0.05) for induction of somatic embryogenesis, as evident by the embryogenic callus formation (Fig. 1E). The maximum callogenic frequency (95.37 ± 2.44%) obtained at 1 mg/l 2,4-D showed the least frequency of embryogenic callus formation (19.44 ± 2.77%). On the contrary, though the callogenic frequency obtained at 4 mg/l 2,4-D was less (67.13 ± 2.82%) compared to the treatment of 1 mg/l 2,4-D, it was more significant for embryogenic callus formation with 58.33 ± 4.81% frequency (Fig. 3A). NAA was significantly productive for somatic embryogenesis at 2 mg/l and 4 mg/l concentrations (Fig. 4A). The MS basal media without any growth regulator (control) was insignificant in forming a non-embryogenic and embryogenic callus. It was observed that the explant (cotyledon) remained unresponsive in the absence of any growth regulator (Fig. 1F). Among all individually tested growth regulators, 4 mg/l 2,4-D proved significantly more efficient for the induction of somatic embryogenesis.
Cream-colored, shiny, translucent, smooth-surfaced, and compact somatic embryos developed through callus formation from the cut edges of the cotyledonary explant. The development of somatic embryos was observed to be initiated by the end of 5 th week. The somatic embryo formed and matured asynchronously through various developmental stages; globular (Fig. 1G), heart (Fig. 1H), torpedo (Fig. 1I), and cotyledonary (Fig. 1J). The treatments with high NAA concentration (individually or in combination) resulted in direct shoot formation.
It was observed that from a less number of globularstaged somatic embryos that were produced from the callus obtained on the surface of the explant, the lesser number of them matured to the cotyledonary-staged somatic embryos. The average number of globular-staged somatic embryos ranged between 1and 5, irrespective of the treatments. The maximum average number of cotyledonarystaged embryos (1.53 ± 0.04) was obtained at 4 mg/l 2,4-D (Fig. 3B). The NAA produced a maximum of 1.12 ± 0.05 cotyledonary-staged somatic embryos at 4 mg/l (Fig. 4B). Combination of 0.1 mg/l BAP with 2 mg/l 2,4-D produced significantly (p < 0.05) higher (2.85 ± 0.04) number of cotyledonary-staged somatic embryos (Table 1). However, this data did not differ significantly from those obtained with the treatment of 0.01 mg/l BAP + 2 mg/l 2,4-D or 0.01 mg/l BAP + 4 mg/l 2,4-D. The combination of two growth regulators, a cytokinin and an auxin, showed an

Somatic embryo conversion to plantlet
The conversion of somatic embryos, developed from cotyledons of P. santalinus to plantlets, was unproductive on the hormonal medium (data not shown). Therefore cotyledonary-staged somatic embryos were transferred to MS basal medium (Fig. 5A, B). The somatic embryos were converted into a complete plantlet with the concurrent development of a shoot and root (Fig. 5C-E). It was also noticed that sometimes, instead of developing into complete plantlets, the matured cotyledonary-staged somatic embryos developed into either shoots or roots. Transferring embryos from individual PGR treatment to basal media did not support somatic embryo conversion. The plantlets were generated only when embryos were transferred from combinations of PGR treatments to basal media, with the highest number of average plantlets (1.30 ± 0.07) obtained at 0.1 mg/l BAP + 2 mg/l 2,4-D (Table 1).

Acclimatization of plantlets
Though a maximum of only 1.30 plantlets were formed from somatic embryo conversion but were successfully established during acclimatization in laboratory conditions and a greenhouse environment. The plantlets produced from the conversion of somatic embryos, generated from all the applied treatments, were acclimatized well and survived 100%. Under laboratory conditions in plastic bottles, the plantlets grew 6-7 cm in height and developed 2-3 leaflets and a thick root (Fig. 5F). After transferring to progressively bigger pots, it was observed that plants also grew progressively in height to 28 cm (Fig. 5G) with increased numbers and sizes of the leaves. Finally, after being transferred to the greenhouse, there observed healthy development of the plants (Fig. 5H).

Histological studies
The histological analysis helped to differentiate between embryogenic and non-embryogenic cells with the former having dense cytoplasm and the latter having large vacuoles (Fig. 6A). Somatic embryos showed the presence of protoderm and accumulation of meristematic cells (Fig. 6B). Globular structures were developed after 5 weeks of induction (Fig. 6C). The transition from globular to heart-staged showed the attachment of embryogenic cells to a suspensor (Fig. 6D). The polar migration of auxin with the organization of embryogenic tissues towards the periphery with a globular embryo at the tip marked the importance of the mechanism called polar-auxin transport (Fig. 6E).

Discussion
The study's observations highlighted the importance of growth regulators and induction period for establishing somatic embryogenesis in P. santalinus. The significance of auxins, especially 2,4-D, was already confirmed for somatic embryogenesis in the case of woody (Mehta et al. 2011;Nunes et al. 2018) and Fabaceous trees (Han and Park 1999;Trigiano et al. 1999;Buendía-González 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 strength 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 their role in the 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 compared to their individual treatments. A similar result has also been reported by Canhoto et al. (2006) in carob, where they observed somatic embryogenesis when BAP and IAA were used in combinations, whereas the individual use of hormones proved ineffective. Auxins and cytokinin play a vital role in regulating 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). The current study used cotyledon explants from germinated immature zygotic embryos to produce 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 the induction of somatic embryogenesis while working with carob. The difference in in-vitro condition or the genotype of the plant and the explant has a significant effect on the successful induction and maintenance of somatic embryogenesis (Canhoto et al. 2006). Gulzar et al. (2020) reported that though with the advancement of technology the difficulty in establishing somatic embryogenesis has been overcomed, most woody species either remain recalcitrant or respond poorly. They also mentioned the incapability of embryonic cells to develop into complete plantlets. Our results with fewer globular-staged embryos, cotyledonary-staged embryos, and plantlets have validated the same. The period for induction of somatic embryogenesis varies in different plant species. Some plants require a short period of induction, as reported by Sharry et al. (2006) while working with a tree species Melia azedarach L. and Chitra Devi and Narmathabai (2011) with a Fabaceae species Desmodium motorium (Houtt.) Merr. On the contrary, others require prolonged induction, as Wang et al. (2003) reported while working with a tree species Areca catechu L. and Canhoto et al. (2006) with a Fabaceae species Ceratonia siliqua L. In the current study, the somatic embryos were obtained after prolonged induction of 12 weeks. Singh and Chand (2003) worked with a Fabaceae plant, Dalbergia sissoo Roxb., and obtained 26.5 average number of somatic embryos after 15 weeks of culture in half-strength MS media.
The success of micropropagation through somatic embryo formation depends on its development and final conversion to plantlets, which remains challenging 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 to complete the morphogenic process. Weaver and Trigiano (1991) worked with a Fabaceae plant, Cladrastis lutea, and reported the lack of somatic embryo conversion to plantlet. The conversion problem might be due to the anomalous shoot apex formation, which 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% embryo conversion using cotyledon as an explant. They used 10% sucrose for the maturation and 2% sucrose for the germination of the matured somatic embryos. A high sucrose concentration before germination improves the process as it signals for biogenesis of stored proteins (Singh and Chand 2003). On the contrary, the high sucrose concentration may inhibit germination due to osmotic shock if present in the germination medium (Verma et al. 2016). Hence, the development of somatic embryos and their successful conversion also depends on various factors like the explant, growth regulators, and duration of the use of growth regulators. 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 the successful conversion of somatic embryos. Lemes da Silva et al. (2021) reported that 18.1 μM 2,4-D + 4.5 μM BAP resulted in maximum somatic embryos and was the only treatment regenerating plantlets after their transfer to basal media.
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 the 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 nonembryonic cells and the attached suspensor. It helps to track the polar movement of the embryonic callus toward the tip (Hasbullah et al. 2007). The attached suspensor with the embryonic cells, a characteristic feature of somatic embryos, has also been shown by Xia et al. (2021) during their work on Masson pine (Pinus massoniana). An early phase of somatic embryo development with dense cytoplasm at the embryonal end with a 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).

Conclusion
In this work, we present the first approach to the regeneration of P. santalinus through somatic embryogenesis. This protocol showed that the red sandalwood could be regenerated from somatic embryos with a maximum embryogenic frequency of 69.44%. However, future studies are required to increase the frequency of somatic embryos formed and convert them into plantlets. Apart from this, molecular approaches through proteomic expressions are needed to optimize the protocol for mass production at a commercial scale and to understand the evolution, structure, and genetic organization of this plant species. In conclusion, the present study can be a step ahead in the large-scale propagation regenerated through somatic embryogenesis to overcome the endangered status of P. santalinus species.