Somatic Embryogenesis and Plantlet Regeneration in Red Sandalwood (Pterocarpus Santalinus)

doi:10.21203/rs.3.rs-2007849/v1

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Somatic Embryogenesis and Plantlet Regeneration in Red Sandalwood (Pterocarpus Santalinus)

https://doi.org/10.21203/rs.3.rs-2007849/v1

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Cotyledonary segments from the germinated immature zygotic embryo were used for somatic embryogenesis of red sandalwood (Pterocarpus santalinus). It was established on Murashige and Skoog (MS) medium containing 5% sucrose and amalgamation of 6-benzylaminopurine (BAP), 2,4-Dichlorophenoxyacetic acid (2,4-D), and α-Naphthaleneacetic acid (NAA). All treatments were responsive for callus induction with the frequency range between 36–97%. The attempts to obtain embryogenesis with 3% sucrose were ineffective since only non-embryonic callus was observed. The maximum frequency of embryogenic induction (69.44%) was obtained in 0.1 mg/l BAP and 2 mg/l 2,4-D; the data was not significantly (p < 0.05) different from the result obtained when 0.1 mg/l BAP and 4 mg/l 2,4-D were used. When explants were treated individually with growth regulators, the maximum (58.33%) frequency was produced by 4 mg/l 2,4-D. The BAP was completely ineffective when used individually. The maximum number of an advanced-stage embryo (2.85) was obtained with treatment 0.1 mg/l BAP and 2 mg/l 2,4-D. The maximum plantlets were developed (1.30) when the cotyledonary-staged embryo from 0.1 mg/l BAP and 2 mg/l 2,4-D were transferred to MS basal medium. The maturation and germination of somatic embryos were challenging, suggesting the need for molecular approaches through proteomic expression for mass production and understanding the evolution, structure, and genetic organization of the plant species. The plantlets obtained were acclimatized and survived in green house condition. Histological observation showed that the cells of the globular embryos had a dense cytoplasm and a long suspensor and used to distinguish between embryonic and non-embryonic cells.

callogenesis

cotyledon

Fabaceae

histology

somatic embryo

The growth regulators, especially 2,4-D, has a significant effect on embryonic potential of cotyledon explant of P. santalinus.

Pterocarpus santalinus is a woody tree of the Fabaceae family, endemic to the Deccan part of the Indian peninsula (Ahmedullah et al. 2019). However, it is cultivated in China, Pakistan, Sri Lanka, and Philippines. It is an endangered species, listed in Endangered A2cd ver 3.1 of IUCN Red List, with an exceptional timber quality. The plant has been smuggled over time, mainly in China, Japan, and Myanmar, and hence cataloged in Appendix II of CITES (UNEP-WCMC 2017; Ahmedullah 2021). The hardwood is 16% saturated with a red-colored dye, known as santalin, used as a coloring agent in 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) (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 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 (Lakshmi Sita et al. 1992; Anuradha and Pullaiah 1999; Arockiasamy et al. 2000; Prakash et al. 2006; Rajeswari and Paliwal 2008; Padmalatha and Prasad 2008; Balaraju et al. 2011; Warakogoda and Subasinghe 2013; Chen et al. 2019). 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, it has also been achieved in many leguminous plants (Singh and Chand 2003; Buendı´a-Gonza´lez et al. 2012; Viji et al. 2012; Kaul et al. 2014; Nolan et al. 2014), including P. marsupium (Husain et al. 2010). To our knowledge, there is no report of somatic embryogenesis in P. santalinus. Recently in vitro morphogenesis responses is reported from cotyledonary explants of immature zygotic embryos of P. santalinus (Chakraborty et al. 2022a) following the analysis of regeneration protocols for micropropagation (Chakraborty et al. 2022b). This study documents the regeneration of plantlets by establishment of somatic embryogenesis in red sandalwood and histological analysis to differentiate embryonic and non-embryonic cells from cotyledon explants.

Plant material and surface sterilization

Immature green-winged pods of P. santalinus were collected from a 15-year-old plant grown in the Andhra Pradesh Red Sandalwood Forest Patch (Latitude N 17^o 53´ 9.955˝, Longitude E 83^o 19´ 42.648˝) (Fig. 1A). The wings were excised from the pods (Fig. 1B), and rinsed under tap water for 10–15 minutes 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 minutes, followed by treatment with 0.1% HgCl₂ for 15 minutes inside Laminar Air Flow (LAF; KEMI, India). Finally, it was rinsed with sterilized double-distilled water 4–5 times for 30 sec to 1 min. The immature zygotic embryos were dissected out by removing the hard outer layer of the pods with the help of a wire cutter (Fig. 1C). It was then placed on MS (Murashige and Skoog, 1962) medium for germination. After 28 days, the cotyledon sections of the size 0.5–1 cm were sliced out from the 5–6 cm height plantlet and used as an explant to induce somatic embryos (Fig. 1D).

Culture media and culture condition

The explants were cultured in test tubes (25×150mm, BOROSIL) containing 15 ml of MS basal media adjusted to pH 5.8 ± 0.2 using 1N NaOH before autoclaving at 121ºC for 15 minutes. The media was added with BAP (6-Benzylaminopurine; PCT0802, HIMEDIA) at 0.01, 0.1, 1.0, and 2.0 mg/l; 2,4-D (2,4-Dichlorophenoxyacetic acid; PCT0825, HIMEDIA) at 0.01, 0.1, 1.0, 2.0, and 4.0 mg/l; NAA (α-Naphthaleneacetic acid; PCT0809, HIMEDIA) at 1.0, 2.0, 4.0, and 8.0 mg/l or combination of all of them. 5% (w/v) sucrose (PCT0607, HIMEDIA) and 1% (w/v) agar powder (PCT0901, HIMEDIA) were added to all induction medium. The basal media without PGR (plant growth regulator) were kept as a control for comparing results. The cotyledons were placed in contact with the culture medium. All the cultures were kept in an air-conditioned tissue culture room at 25 ± 2℃, 60–65% relative humidity, and a 16/8 hr (light/dark) photoperiod under 40 W cool white fluorescent tube at 70 µM m^− 2s^− 1 lights. Each treatment were replicated thrice with 12 cotyledon segments per replication, which were performed from July 2019 to January 2022, and the results were recorded after 12 weeks of culture.

Cotyledonary-staged somatic embryos were transferred to full-strength MS basal media with 3% (w/v) sucrose and 1% (w/v) agar powder for germination and kept in air-conditioned tissue culture room maintained at, 25 ± 2℃, 16/8 hr (light/dark) photoperiod receiving 70 µMm^− 2s^− 1 with 40 W cool white fluorescent tube lights. Well-developed plantlets were shifted to a plastic glass containing garden soil and vermiculite in a 1:1 (w/w) ratio after treating with fungicide (Bavistine) for 2–4 min. The glass was covered with another plastic glass to maintain humidity in the culture room for 4–5 weeks with intermittent watering. The well-established plantlets under culture room conditions were processed for acclimatization under laboratory condition in shade for another 2–3 weeks. The plantlets were finally transferred to green house for further hardening.

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) average number of advanced-stage embryo, and (ii) average number of plantlets per treatment. The response parameters were calculated as:

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

Histological studies

Embryonic tissue was observed by staining with 2% (w/v) acetocarmine for 30 sec, by placed it on a glass slide. The staining solution was washed with distilled water before mounting with a cover slip. The microscopy was performed under light microscopy (CH20 i, Olympus, Japan).

Acclimatization

Acclimatization of regenerated somatic plantlets were performed following the step described earlier (Chakraborty et al. 2022a & b).

Statistical analysis

The results for all the experiments were statistically analyzed using SPSS (IBM, version 19.0). Mean with the standard error was calculated with Microsoft Excel (2016 version) and presented in tabular form. The average callogenic frequency, embryogenic frequency, advanced-stage embryo per treatment, and plantlets generated per treatment were subjected to analysis of variance (ANOVA). Significant differences among 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, 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. 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, the callogenic frequency obtained at 4 mg/l 2,4-D was 67.13 ± 2.82%, but it was more significant for embryogenic callus formation with 58.33 ± 4.81% frequency (Fig. 3A and 3B). NAA was found significantly productive for somatic embryogenesis at 2 mg/l and 4 mg/l concentration (Fig. 4A and 4B). The MS basal media without any growth regulator (control) was not significant in forming both non-embryogenic or embryogenic callus. Among all individually tested growth regulators, 4 mg/l 2,4-D proved to be significantly more efficient for induction of somatic embryogenesis.

The combination of cytokinin BAP with auxin 2,4-D or NAA was also tested for somatic embryogenesis induction. The maximum callogenic frequency (82.41 ± 4.04%) was obtained at 0.1 mg/l BAP + 2 mg/l 2,4-D (Table 1). The highest embryogenic frequency (69.44 ± 2.77%) was obtained at 0.1 mg/l BAP + 2 mg/l 2,4-D which do not differ significantly from the result obtained at 0.1 mg/l BAP + 4 mg/l 2,4-D (Table 1). Independent of the concentrations of BAP used (0.01 and 0.1 mg/l), the presence of 2,4-D (1, 2, and 4 mg/l) or NAA (2 and 4 mg/l) significantly (p < 0.05) increased the frequencies of embryogenic induction, except for the combination 0.01 mg/l BAP + 4 mg/l 2,4-D (Table 1). Combining the three growth regulators significantly (p < 0.05) decreased the embryogenic frequency. The individual and combination treatments were responsive for callus induction with the frequency range between 36–97%. The combination of BAP with either 2,4-D or NAA was more efficient for somatic embryogenesis induction than their individual or triple combination treatment.

Table 1

Influence of combination of BAP × 2,4-D, BAP × NAA, and BAP × 2,4-D × NAA on frequency of callogenesis and somatic embryogenesis, somatic embryo maturation and conversion to plantlets
Growth regulators (mg/l)			Frequency of callogenesis (%)	Frequency of embryogenesis (%)	Average No. of Advanced stage embryos per treatments	Average No. of plantlets per treatments (after transferring to MS basal)
BAP	2,4-D	NAA	Frequency of callogenesis (%)	Frequency of embryogenesis (%)	Average No. of Advanced stage embryos per treatments
0.00	0.00	0.00	0.00 ± 0.00ⁱ	0.00 ± 0.00^h	0.00 ± 0.00^h	0.00 ± 0.00^h
0.01	1.00	0.00	73.15 ± 0.93^bc	50.00 ± 4.80^c	1.83 ± 0.05^c	0.85 ± 0.02^ef
0.01	2.00	0.00	71.30 ± 2.45^bc	61.11 ± 2.78^ab	2.62 ± 0.10^a	1.08 ± 0.05^bcd
0.01	4.00	0.00	69.45 ± 2.78^bc	52.77 ± 2.77^bc	2.84 ± 0.09^a	1.24 ± 0.05^ab
0.01	0.00	2.00	50.00 ± 3.21^def	36.11 ± 2.78^e	1.00 ± 0.08^f	0.92 ± 0.12^de
0.01	0.00	4.00	60.19 ± 3.34^cd	47.22 ± 2.77^cd	1.84 ± 0.08^c	0.94 ± 0.04^cde
0.01	1.00	2.00	63.89 ± 2.78^cd	38.89 ± 2.78^de	2.22 ± 0.10^b	1.07 ± 0.06^bcd
0.01	2.00	2.00	58.33 ± 4.81^cd	13.89 ± 2.78^g	0.91 ± 0.03^fg	0.48 ± 0.04^g
0.01	1.00	4.00	72.22 ± 2.77^bc	36.11 ± 2.78^e	1.65 ± 0.06^cd	0.68 ± 0.04^f
0.01	2.00	4.00	44.44 ± 2.77^fgh	11.11 ± 2.78^g	0.74 ± 0.04^g	0.33 ± 0.03^g
0.10	1.00	0.00	57.41 ± 4.90^cd	50.00 ± 4.80^ab	2.38 ± 0.09^b	1.01 ± 0.07^cde
0.10	2.00	0.00	82.41 ± 4.04^a	69.44 ± 2.77^a	2.85 ± 0.04^a	1.30 ± 0.07^a
0.10	4.00	0.00	75.00 ± 4.81^bc	63.89 ± 2.78^a	2.18 ± 0.04^b	1.13 ± 0.06^abc
0.10	0.00	2.00	75.00 ± 4.81^bc	52.77 ± 5.55^bc	1.54 ± 0.10^d	0.68 ± 0.09^f
0.10	0.00	4.00	72.22 ± 2.78^bc	61.11 ± 2.78^ab	1.11 ± 0.06^ef	0.74 ± 0.04^f
0.10	1.00	2.00	41.66 ± 4.81^gh	25.00 ± 4.80^f	2.13 ± 0.04^b	0.69 ± 0.04^f
0.10	2.00	2.00	47.22 ± 2.77^efg	0.00 ± 0.00^h	0.00 ± 0.00^h	0.00 ± 0.00^h
0.10	1.00	4.00	55.55 ± 2.77^cde	19.44 ± 2.77^fg	1.28 ± 0.15^e	0.32 ± 0.03^g
0.10	2.00	4.00	36.11 ± 2.77^h	0.00 ± 0.00^h	0.00 ± 0.00^h	0.00 ± 0.00^h
Standard errors of means are indicated. Values followed by the same letter are not significantly different by Duncan multiple comparison test (p ≤ 0.05).

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 5th week. Somatic embryo formed and matured asynchronously through various developmental stages; globular (Fig. 1E), heart (Fig. 1F), torpedo (Fig. 1G), and cotyledonary (Fig. 1H). Some treatments, particularly with higher NAA concentration (individually or in combination), resulted in direct shoot formation. The number of somatic embryos (globular-staged) ranged between 1–50 irrespective of the treatments (data not shown).

It was observed that lesser number of globular-staged somatic embryos matured to advanced-staged somatic embryos. The maximum average number of an advanced-stage embryo (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 advanced-stage embryo 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 advanced-stage somatic embryos (Table 1). However, this data did not differ significantly from the data obtained with 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 a synergistic effect for the induction and development of somatic embryos.

Our study witnessed the challenges in developmental advancement at various stages from the globular to cotyledonary phase. Efforts to overcome the challenges were unproductive since either the embryos remained non-responsive or turned dark-brown after 2–3 weeks of treatment (data not shown). Though the frequency of somatic embryogenesis obtained was satisfactorily, we observed a reduced conversion to advanced-stage somatic embryos.

Somatic embryo conversion to plantlet

The conversion of somatic embryos of P. santalinus to plantlets were unproductive on the hormonal medium. Therefore cotyledonary-staged embryos were transferred to MS basal medium (Fig. 5A). The somatic embryos were converted into a complete plantlet with concurrent development of shoot and root (Fig. 5B, 5C, and 5D). It was also noticed that sometimes, instead of developing into complete plantlet, the matured embryos only developed 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 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

The regenerated somatic plantlets were hardened and acclimatized following the procedure described in Chakraborty et al. (2022a & b). The plantlets formed from somatic embryos were acclimatized well and 100% survived in the laboratory and the greenhouse conditions (Fig. 5E and 5F).

Histological studies

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-stage showed embryogenic cells attached with a suspensor (Fig. 6D). The polar migration of auxin with organization of embryogenic tissues towards periphery with globular embryo at the tip marked the importance of the mechanism polar-auxin transport (Fig. 6E).

The observations of the study highlighted the importance of growth regulators and induction period for 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 embryo 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). We worked on cotyledon explants from germinated immature zygotic embryo to produce somatic embryos. Other mature explants resulted in formation of non-embryonic friable callus (data not shown). Canhoto et al. (2006) showed the importance of explant for induction of somatic embryogenesis while working with carob. Bayarmaa et al. (2018) and Ebrahimi et al. (2018) used cotyledon explant while working with an endangered medicinal plant, Zygophyllum potaninii and Kelussia odoratissima Mozaff. respectively. Contrary, Nolan et al. (2014) worked with Medicago truncatula and established somatic embryogenesis from mature leaf explant. Similarly, Xu et al. (2019) developed somatic embryos with root, stem, and leaf explant of Ranunculus sceleratus plant. Da Silva et al. (2018) also proved leaves to be an appropriate explant to develop the somatic embryo in case of Byrsonima intermedia. The difference of in-vitro condition or the genotype of the plant and explant could result in such disparity (Canhoto et al. 2006). Gulzar et al. (2020) explained the rationale behind the recalcitrant behavior of woody species. They also mentioned the incapability of embryonic cells to develop into complete plantlets, the same validated by our results with lesser number of the advanced-stage embryo and the plantlets.

The period for induction of somatic embryogenesis varies in different plant species. Some plants require a specific period of induction (Sharry et al. 2006), whereas others require prolonged induction (Wang et al. 2003). We obtained somatic embryos after prolonged induction of 12 weeks. The work carried out with an induction period of 7, 14, 21, 28, 35, and 42 days resulted in non-embryogenic callus (data not shown). Sucrose is also an important factor for the induction of somatic embryogenesis because it acts as osmotic stress (Verma et al. 2016). We worked with 5% and 3% sucrose, but 3% sucrose resulted in non-embryogenic callus (data not shown). Our observation is 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, posttranscriptional, and posttranslational processes. Sucrose plays a role in enhancing the expression of the genes for 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). We encountered the same challenge 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 a non-complete maturation and capacity in completing the morphogenetic process. Similar observations have 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, without the need for a second rooting treatment. The hardening procedure described by Chakraborty et al. (2022a & b) for P. santalinus proved cent per cent successful for the acclimatization applying a mixture of farmyard manure and vermiculites in a ratio of 1:1 or 1:2 for plantlets regenerated through of somatic embryogenesis. The growth of somatic plantlets during hardening and the acclimatization process was slow but grew up to 15–18 cm shoot length during 7–8 weeks of transfer to the soil. The slow growth of plantlets during the whole of the acclimatization process was also reported by Chakraborty et al. (2022a & b). Similar success rate was obtained by maintaining the relative humidity from 85 to 65% during red sandalwood plantlets acclimatization by Prakash et al. (2006). While, Rajeswari and Paliwal (2008) reported only 95% success rate during the acclimatization of plantlets in coarse sand, clay, and farmyard manure in a ratio of 1:1:1 mixture.

Histological analysis showed the presence of a dense cytoplasm within the parent cell wall, a characteristic feature of somatic embryos. The same characteristics were 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 Cunninghamia lanceolata of zygotic embryo (Hu et al. 2017). 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). The histological analysis helped to differentiate between embryogenic and non-embryogenic callus formed simultaneously from the same explant.

The obtained result is the first approach for regeneration of P. santalinus through somatic embryogenesis that can prove to be an efficient protocol for the propagation of the species. However, further studies are required to overcome the challenges of embryo maturation and conversion into plantlets. Molecular approaches through proteomic expression 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.

2,4-D: 2,4-Dichlorophenoxyacetic acid

BAP: 6-Benzylaminopurine

MS: Murashige and Skoog

NAA: α-Naphthaleneacetic acid

PGR: Plant growth regulator

ACKNOWLEDGEMENT

The financial support provided by SCIENCE & ENGINEERING RESEARCH BOARD (SERB), Department of Science and Technology, Government of India for the major research project under a core research grant to Dr. Nasim Akhtar (Principal Investigator) and Dr. K. Viswanatha Chaitanya (Co-investigator) (sanction order no. CRG/2018/000517 dated 24.6.2019) is gratefully acknowledged.

AUTHOR'S CONTRIBUTION

The manuscript's concept, layout, and design are conceived by the corresponding author Dr. Nasim Akhtar. The whole manuscript, including the table and text, is developed, written and revised by the first author Tanushree Chakraborty. The manuscript was reviewed and edited several times by authors Dr. K. Viswanatha Chaitanya and Dr. Nasim Akhtar.

DATA AVAILABILITY STATEMENT

Data will be made available by the corresponding author on reasonable reason request.

CONFLICT OF INTEREST

All the authors declared that there is no conflict of interest concerning any part of the manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

CONSENT FOR PUBLICATION

Not applicable.

COMPETING INTERESTS

The authors declare that they have no competing interests.

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Somatic Embryogenesis and Plantlet Regeneration in Red Sandalwood (Pterocarpus Santalinus)

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Abstract

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Key Message

Introduction

Material And Methods

Plant material and surface sterilization

Culture media and culture condition

Growth measurement

Histological studies

Acclimatization

Statistical analysis

Results

Somatic embryogenesis induction

Somatic embryo conversion to plantlet

Acclimatization of plantlets

Histological studies

Discussion

Conclusion

Abbreviations

Declarations

References

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