Introduction

The genus Artemisia (family Asteraceae) is a source of numerous phytochemical compounds (Pandey and Singh 2017; Bisht et al. 2021). Some species of the genus are vulnerable due to restricted distribution, numerous anthropogenic activities, and non-judicious collection to meet the increasing demands of various pharmaceutical industries (Sainz et al. 2017). Artemisia maritima L., one such species of this genus, has restricted distribution and is found in only some areas of North Western India including Kashmir, Kurram, Kishtwar, and Gurez (Parihar et al. 2011; Bhagat and Singh 1989; Hooker 1882). The plant is medicinally important and contains an array of phytochemicals. Artemisinin, an important antimalarial drug, has also been reported to be present in A. maritima (Singh et al. 2021). This plant possesses antihelminthic (Irum et al. 2015), pesticidal (Walia et al. 2019), anticancer (Qadir et al. 2019), anticytotoxicity (Qadir et al. 2019), antibacterial properties (Stappen et al. 2014), and antiplasmodial activities (Ene et al. 2009). According to ENVIS (2003) center on medicinal plants, with reference to Jammu and Kashmir (India), A. maritima is categorized as vulnerable. The populations of A. maritima are dwindling due to various anthropogenic activities although certain intrinsic factors also contribute to its dwindling populations (Bharti et al. 2019). So, these threats and causes advocate for in vitro germplasm conservation of this medicinally important species.

In vitro propagation is a powerful tool for germplasm conservation and mass multiplication (Cardoso and Da Silva 2013; Nabi et al. 2021; Nazir et al. 2021). This technique has been used for regeneration in many medicinal and aromatic plants, including several species of Artemisia (Ali et al. 2017; Al-Khayri 2018; Deepa and Thomas 2020). Micropropagation is a technique utilized for rapid multiplication, production of disease-free, uniform, and genetically stable progenies, and production of plant secondary metabolites (Zadoks 2013; Koul et al. 2017; Adhikary et al. 2021). It has various benefits over the conventional ways of propagation, including independent of season, small space requirement, and high-quality production (Kulus 2015).

Plant regeneration via callus formation often gives rise to somaclonal variations, which is a great challenge for the production of true-to-type plants. The somaclonal variants may arise due to epigenetic changes or permanent genetic changes (Ali et al. 2017; Ghosh et al. 2021). Variations in plants can be analyzed by various molecular markers such as amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSR), random amplification of polymorphic DNA (RAPD), start codon targeted (ScoT) polymorphism, and multiomics approaches (Butiuc-Keul et al. 2016). These methods are quite expensive and laborious and require various primers. The determination of DNA content by flow cytometry (FCM) requires a lesser amount of biological sample, is inexpensive, and can be achieved at the in vitro multiplication stage (Ochatt 2008; Ochatt et al. 2013; Miler et al. 2020). Since there are no reports available in the literature on micropropagation, somatic embryogenesis, and genome size analysis in A. maritima, this study aimed at designing an efficient reproducible micropropagation protocol for A. maritima in order to conserve its germplasm and development of true-to-types.

Materials and Methods

Callogenesis and Direct Plant Regeneration

A. maritima L. plants were collected from Gurez, Bandipora, Jammu and Kashmir, India (34.38° N, 74.43° E), 2393 m above sea level (Supplementary File 1), identified under voucher no. 3146-(KASH), and established at KUBG (Kashmir University Botanical Garden, Srinagar, India). The explants (leaf and stem) were collected in a beaker containing water, washed under running tap water for about 30 min, surface sterilized by rinsing with labolene (Himedia, Maharashtra, India) for 10 min, and thoroughly washed under running tap water for the next 20 min. For about 8 min, under a laminar air flow hood, the explants were subjected to surface sterilization with 2% sodium hypochlorite (v/v) (Himedia), and washed (3 times) with autoclaved double distilled water. The explant was cut to make an appropriate size (5 to 15 mm) by trimming the dead tissue and was placed on a sterile Murashige and Skoog (MS; Murashige and Skoog 1962) medium containing 3% sucrose (w/v) (Himedia) and solidified with 0.8% agar (w/v) (Himedia). The MS medium was fortified with different plant growth regulators (PGRs) (purchased from Sigma-Aldrich, St. Louis, MO) as mentioned in Table 1. These cultures were kept at 25 ± 2 °C with 16 h of photoperiod and subcultured after every 2 wk. The treatments were performed with three replicates (1 replicate represents 3 callus pieces per test tube).

Table 1. Concentration of different plant growth regulators (PGRs) fortified with Murashige and Skoog medium (2,4-D, 2,4-dichlorophenoxyacetic acid; BAP, 6-benzylaminopurine; IAA, indole-3-acetic acid; KN, kinetin; NAA, α-naphthaleneacetic acid; TDZ, thidiazuron)
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Somatic Embryogenesis and Indirect Shoot Regeneration

Somatic embryogenesis was induced after successful inoculation of the green callus obtained from stem explant on MS medium supplemented with 0.5 mg L−1 thidiazuron (TDZ), 1.0 and 1.5 mg L−1 indole-3-butyric acid (IBA), and 0.5 mg L−1 α-naphthaleneacetic acid (NAA). Indirect shoots were observed after 2 wk of callus subculture on MS medium supplemented with different concentrations of 0.5 to 2.0 mg L−1 NAA and 0.5 to 2.0 mg L−1 IBA and their cumulative concentrations. The temperature of 25 ± 2 °C and 55 to 70% relative humidity were sustained for the cultures. All the experiments were repeated at least thrice with 12 replicates for each experiment (1 replicate represents 3 callus pieces per test tube).

Hardening and Acclimatization

After washing, plants with roots were transferred to jiffy pots filled with vermiculite and maintained under 28 ± 3 °C with a relative humidity of 75 to 80%. Meanwhile, these were transferred to plastic pots containing soil, sand, and manure in the ratio of 1:1:1 for acclimatization. After 2 wk, the acclimatized plants were relocated to a greenhouse under controlled photoperiod parameters.

SEM Analysis

Embryogeneic callus was fixed for 24 h at 4 °C in 2.0% glutaraldehyde, 2.0% formaldehyde, at pH 6.8 followed by washing with a buffer and fixed with 1% osmium tetraoxide, and dehydrating in ethanol graded series and finally coating with gold palladium. Lastly, samples were then photographed in EVO 18 (Carl Zeiss, Bangalore, India) scanning electron microscope functioning at 15 to 25 kV.

Histology

The organogenic callus was fixed in FAA (formalin, glacial acetic acid, 70% ethanol (5:5:90), followed by dehydration in graded ethanol series and implanted in paraffin (Johansen 1940). Sections of about 8 µm were cut with the rotatory microtome (Sigma-Aldrich, St. Louis, MO). The sections were dewaxed and stained with 5% hematoxylin and 2% eosin and finally mounted on glass slides. These sections were then visualized and microphotographed (Carl Zeiss).

Flow Cytometry

In this study, genome size analysis was performed for field-grown plants, plants regenerated via direct organogenesis, and somatic embryo–derived plantlets. For this purpose, three randomly selected plants were analyzed from each experimental setup of in vitro grown Artemisia (plants regenerated via direct organogenesis, and somatic embryogenesis), and from in vivo grown control group. Plant material of Artemisia maritima nearly 1.0 cm2 and reference standard [Pisum sativum (2C DNA = 9.56 pg)] were meshed in 0.5 mL Otto buffer (0.3% citric acid monohydrate, 0.05% NP-40), 50 µg mL−1 propidium iodide, and 100 µg mL−1 Rnase (Sigma-Aldrich). The genomic samples prepared were sieved through a 100 μm mesh sieve, before being examined through (CFM) BD FACS Calibur (BD Biosciences, San Jose, CA) flow cytometer (Dolezel et al. 2007). Nuclear DNA content (2C) of A. maritima was calculated using the below formula:

$$2\mathrm{C DNA content of} A. maritima \mathrm{L}.=9.56\times \frac{\mathrm{Mean position ofG}0/\mathrm{G}1\mathrm{peak of} A.maritma L.}{\mathrm{Mean position ofG}0/\mathrm{G}1\mathrm{peak of }Pisum sativum}$$

Statistical Analysis

All the treatments were performed in replicates (n = 3), and the presented data was expressed as mean ± standard deviations (SD). Significant differences between the mean values were assessed by one-way ANOVA followed by post hoc Duncan’s multiple range tests (DMRT) at p ≤ 0.05, using SPSS software (version 22, IBM Armonk, New York).

Results

Callus Induction and Proliferation

Callus induction was witnessed in stem and leaf explants inoculated on MS media amended with varying concentrations of auxins [2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), IBA, and NAA] and cytokinins [6-benzylaminopurine (BAP) and kinetin (KN)]. The best callus induction was observed on 2.5 mg L−1 2,4-D in combination with 1.5 mg L−1 BAP. The percent response of stem varied from 38.65% (0.5 mg L−1 2,4-D and 0.5 mg L−1 BAP) to 81.32% (2.5 mg L−1 2,4-D and 1.5 mg L−1 BAP) (Fig. 1a to d). Similarly, in the case of leaf explant, the percent response varied from 30.64% (0.5 mg L−1 IAA) to 81.26% (1.5 mg L−1 2,4-D and 1.0 mg L−1 BAP) (Fig. 1e to h) (Table 2). The color and texture of callus varied among different growth regulators (Fig. 1a to h). After 4 wk, callus was subcultured on different concentrations of BAP, NAA, and KN (0.5 to 3.5 mg L−1). Among different concentrations, the best proliferation was obtained on MS media fortified with NAA and KN (each 1.0 mg L−1) with a fresh weight of 4.02 ± 0.38 g from stem-originated callus and 3.96 ± 0.35 g from leaf-originated callus, which were significantly different at p ≤ 0.05 according to DMRT (Table 3). Therefore, stem-originated callus proliferated better in comparison to leaf-originated callus. The maximum callus induction frequency and proliferation thus resulted from the cumulative effect of growth hormones.

Figure 1.
figure 1

Callus induction and proliferation in stem and leaf explants of Artemisia maritima L. (a) Brown callus (stem explant) obtained in Murashige and Skoog (MS) medium amended with 2.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D). (b) Brown callus from leaf explant (MS medium amended with 2.0 mg L−1 2,4-D). (c) White yellowish callus from stem (MS medium added with 2.0 mg L−1 α-naphthaleneacetic acid (NAA)). (d) Callus proliferation. (e) Green callus from leaf explant (MS medium amended with 1.5 mg L−1 2,4-D and 1.0 mg L−1 6-benzylaminopurine (BAP)). (f) green callus from stem explant showing regenerated leaves (MS medium amended with 2.5 mg L−1 2,4-D and 1.5 mg L−1 BAP). (g–h) callus proliferation (MS medium amended with 1.0 mg L−1 NAA and 1.0 mg L−1 kinetin (KN)) (scale bars a–h: 0.5 cm).

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Table 2. Callus induction frequency (%), callus texture, and color in stem and leaf explants of Artemisia maritima L. grown in Murashige and Skoog medium amended with different concentrations of plant growth regulators (PGRs) with three replicates for each treatment (1 replicate = 3 callus pieces per test tube) (2,4-D, 2,4-dichlorophenoxyacetic acid; BAP, 6-benzylaminopurine; IAA, indole-3-acetic acid; KN, kinetin; NAA, α-naphthaleneacetic acid)
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Table 3. Callus proliferation in stem and leaf explants of Artemisia maritima L. in terms of fresh weight (g), after 4 wk of inoculation, under different plant growth regulators (PGRs) with three replicates for each treatment (1 replicate = 3 callus pieces per test tube) (BAP, 6-benzylaminopurine; NAA, α-naphthaleneacetic acid; KN, kinetin)
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Shoot Regeneration

The nodal and microshoot tips were aseptically inoculated on MS media amended with 0.5 to 2.5 mg L−1 BAP, 0.5 to 1.5 mg L−1 NAA, and 0.5 to 1.5 mg L−1 TDZ. The best direct shoot regeneration was obtained on 1.0 mg L−1 BAP in combination with 0.5 mg L−1 NAA with 85.25% induction frequency on nodal explant and 90.26% induction frequency on microshoot tips (Table 4). The mean number of shoots per node varied among different concentrations of growth hormones. The maximum number of shoots was noticed on 1.5 mg L−1 BAP in combination with 1.0 mg L−1 NAA. The mean number of shoots was 85.24 ± 3.67 on nodal segments and 88.34 ± 3.82 on microshoot tips after 4 wk of inoculation (Table 4) (Fig. 2a to d).

Table 4. Direct shoot regeneration induction frequency (%) using nodal and microshoot tips as explants in Artemisia maritima L., after 3 wk of inoculation, using different plant growth regulators (PGRs) with three replicates for each treatment (1 replicate = 3 callus pieces per test tube) (BAP, 6-benzylaminopurine; NAA, α-naphthaleneacetic acid; TDZ, thidiazuron)
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Figure 2.
figure 2

Direct shoot regeneration from nodal and microshoot tip explants of Artemisia maritima L. (a–b) Nodal shoot regeneration in Murashige and Skoog (MS) medium amended with 1.0 mg L−1 6-benzylaminopurine (BAP) and 0.5 mg L−1 α-naphthaleneacetic acid (NAA). (c–d) Regeneration from microshoot tip in 1.0 mg L−1 BAP and 0.5 mg L−1 NAA added MS medium (scale bars a–d: 0.5 cm).

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Indirect Shoot Regeneration

After 3 wk of inoculation, a well-established callus resulted in indirect shoot regeneration on MS media amended with 0.5 mg L−1 TDZ, 0.5 mg L−1 NAA, and 1.0 to 1.5 mg L−1 IBA (Fig. 3a to d). The maximum in vitro indirect shoot regeneration induction was obtained on 0.5 mg L−1 TDZ in combination with 1.5 mg L−1 IBA with 80.36% induction frequency and 28.71 ± 0.51 mean number of shoots (Table 5).

Figure 3.
figure 3

Indirect shoot regeneration via callus formation in Artemisia maritima L. (a) Early shoot bud initiation. (b) Little elongation of shoot. (c) Multiple shoot regeneration. (d) Fully developed plant on rooting media (scale bars a–c: 0.5 cm, d: 1 cm).

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Table 5. Indirect shoot induction frequency (%) from callus (derived from 2.0 mg L−1 NAA and 1.5 mg L−1 KN; stem explant) in Artemisia maritima L., after 3 wk of subculturing, using different plant growth regulators (PGRs) with three replicates for each treatment (1 replicate = 3 callus pieces per test tube) (IBA, indole-3-butyric acid; NAA, α-naphthaleneacetic acid; TDZ, thidiazuron; KN, kinetin)
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Rooting

The in vitro cultured shoots were transferred to MS media amended with varying concentrations of 0.5 to 1.5 mg L−1 NAA and 0.5 to 2.0 mg L−1 IBA. The higher root formation frequency was observed on 1.5 mg L−1 NAA (74.36%) with 14.82 ± 0.70 mean number of roots (Table 6). However, no root formation was observed on IAA which instead resulted in callus formation below the shoot.

Table 6. Effect of plant growth regulators (PGRs), NAA, and IBA, on rooting of in vitro regenerated shoots derived from direct/indirect organogenesis of Artemisia martima L. with three replicates for each treatment (1 replicate = 3 callus pieces per test tube) (NAA, α-naphthaleneacetic acid; IBA, indole-3-butyric acid)
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Somatic Embryogenesis

The callus obtained from 2,4-D (brown) and NAA (light yellowish white) failed to form the somatic embryos (SEs) while the callus obtained on BAP and KN resulted in the SE formation (Fig. 4a to f) when placed on MS media amended with 0.5 to 1.0 mg L−1 TDZ and 1.5, 2.0, and 2.5 mg L−1 IBA (Table 7). The germination of SEs was observed on MS media amended with 1.0 to 3.5 mg L−1 BAP and 0.5 to 2.0 mg L−1 NAA (Fig. 4a to h) (Table 8).

Figure 4.
figure 4

Various stages of somatic embyogenesis in Artemisia maritima L. grown in Murashige and Skoog medium. (a) Granular embryogenic callus. (b–c) Globular stage. (d–e) Heart-shaped stage. (f)Germinating embryos with emerging shoot buds (arrows). (g–h) Somatic embryos isolated from embryogenic callus in various stages, radicle and emerging shoot (arrows) (scale bars a–h: 0.5 cm).

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Table 7. Somatic embryogenesis induction frequency (%) in stem explant–derived callus (grown in 3.5 mg L−1 BAP) of Artemisia maritima L. cultured in Murashige and Skoog medium amended with plant growth regulators (PGRs) TDZ and IBA with three replicates for each treatment (1 replicate = 3 callus pieces per test tube) (BAP, 6-benzylaminopurine; IBA, indole-3-butyric acid; TDZ, thidiazuron)
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Table 8. Somatic embryos germination frequency (%) in stem explant–derived callus (grown in 1.0 mg L−1 thidiazuron (TDZ) in combination with 2.5 mg L−1 indole-3-butyric acid (IBA)) of Artemisia maritima L. on Murashige and Skoog medium amended with different concentrations of the plant growth regulators (PGRs) BAP (6-benzylaminopurine) and NAA (α-naphthaleneacetic acid)
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Acclimatization of Plantlets

The plantlets were successfully acclimatized under greenhouse conditions (Fig. 5) following Chandra et al. (2010). The plants were healthy and true-to-type showing a 75% of survival rate. No phenotypic variation was noticed between in vivo grown and regenerated plants.

Figure 5.
figure 5

Rooting induction, hardening, and acclimatization in Artemisia maritima L. (a–b) In vitro root induction in shoots grown in Murashige and Skoog medium amended with 1.5 mg L−1 α-naphthaleneacetic acid, after 4 wk of culture. (c) Plant acclimatization in pots (scale bars a–b: 0.5 cm, c: 2.0 cm).

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Histology

Histological examination of differentiated shoot from callus tissue showed various developmental stages of apical meristem having dome-shaped apical meristem with early leaf primordia (Supplementary File 2). A shoot apex partially covered with two leaf primordia was witnessed at advanced stages. A zone of compact meristematic tissue was detected close to the apex. As shown in Supplementary File 2, embryogenic tissues were observed, which were composed of meristematic cells with dense cytoplasm and prominent nuclei. Subsequently, embryonic cells continued to differentiate, and globular embryos gradually formed after 8 wk (Supplementary File 2).

SEM Analysis

SEM observations revealed the external surface of the embryogenic callus with irregular and frequent protuberances. The emergence of globular SEs from the surface of the embryogenic callus was confirmed by SEM micrographs. The globular SEs then developed through later stages (Supplementary File 2). After 3 wk of callus subculture, the adventitious shoot primordia originated.

Genetic Stability and Genome Size Analysis

The FCM was used to quantify 2C DNA content in order to assess the genetic stability of field-grown and in vitro regenerated plantlets of A. maritima L. For FCM, leaf samples were chopped to obtain nuclear suspensions of field-grown (in vivo) and in vitro regenerated plants (direct organogenesis and somatic embryo–derived). The histograms obtained from FCM analysis confirmed the DNA content (Supplementary File 3). The field-grown plants of A. maritima L. were estimated to contain 14.89 pg of 2C DNA. The 2C DNA content of direct organogenesis derived (14.61 pg) and somatic embryo–derived (14.37 pg) regenerants depicted similarity in their genome size with the field-grown plants (Table 9). Hence, in the present protocol, the obtained regenerants maintained their genetic stability and showed no change in genome size.

Table 9. 2C DNA content [in picograms (pg)] in in vivo and in vitro derived plantlet of Artemisia maritima L. as detected by flow cytometry
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Discussion

In this study, a novel and well-established protocol was developed for callus induction, somatic embryogenesis, and in vitro regeneration of A. maritima L. The callus induction response from leaf and stem explants was observed on almost all concentrations of different growth hormones like 2,4-D, NAA, and BAP. Similar results were also found in Artemisia annua in which callogenesis was observed on all combinations of BAP, NAA, and 2,4-D (Zayova et al. 2020). In this study, the maximum callus induction frequency was observed on 2.5 mg L−1 2,4-D and minimum frequency on 1.5 mg L−1 BAP. Similar observations were noted in A. annua (Zayova et al. 2020). Callus has a variable appearance in texture and shape (Mohajer et al. 2012; Sikdar et al. 2012). Also, in the present study, the color, texture, and embryogenic potential of callus varied with the use of different growth hormones (Table 2; Fig. 1a to h). In the present study, brown, friable, non-embryogenic callus was observed on 2,4-D, while green, hard, and embryogenic callus was seen in plants cultured on BAP and light yellowish white friable and non-embryogenic callus was observed in NAA, on both leaf and stem explants. Aslam et al. (2006) noted similar observations in Artemisia scoparia. The brown and yellowish white callus failed to grow after two subcultures in comparison to the green callus obtained from stem explant while the green callus obtained from the leaf explant also showed browning after two subcultures. The fresh weight of callus after 4 wk of subculturing on different media also varied. The shoot regeneration from callus was observed on TDZ, NAA, and IBA. Similar results were observed by Boo et al. (2015) in Aster scaber in which shoot regeneration from callus was observed on 5 to 25 μM NAA and 0.05 to 25 μM BAP.

Direct shoot regeneration was observed on different concentrations of BAP, TDZ, and NAA using nodal and microshoot tips as explants. The microshoot tips were more responsive as compared to nodal segments. The maximum shoot induction was detected on 1.0 mg L−1 BAP in combination with 0.5 mg L−1 NAA. In Artemisia spicegera, in vitro shoots were observed on 0.5 mg L−1 NAA and 0.5 mg L−1 BAP (Ghorbani et al. 2021). The present study’s results resembled the observations of Lualon et al. (2008) in which shoot regeneration was also observed on BAP that was found to be maximum on 0.1 mg L−1 TDZ in combination with 0.05 mg L−1 NAA and 1.0 mg L−1 BAP. Similar observations were also found by Dangash et al. (2015) on media amended with 1.5 mg L−1 BAP and 0.05 mg L−1 NAA. In Alocosia longiloba, the maximum number of shoots was observed on 3.0 mg L−1 BAP (Abdulhafiz et al. 2020).

Rooting was induced from in vitro regenerated plants using IBA and NAA. The highest root induction frequency was observed on 1.5 mg L−1 NAA. Ghorbani et al. (2021) also noted the rooting on 1.0 mg L−1 NAA and 1.0 mg L−1 IBA. NAA, IBA, and IAA were found to be potent root inducers in Artemisia nilagirica var. nilagirica (Shinde et al. 2016). IBA (2.4, 4.9, 9.8 μM) was also assessed for in vitro rooting in Artemisia annua (Wetzstein et al. 2018). In the present study, concentrations of 0.5 to 4.0 mg L−1 of IAA were used for rooting but no roots were observed on IAA. The present study’s results were in similarity with Jogam et al. (2020) where rooting on regenerated Artemisia vulgaris shoots was observed on MS media augmented with 1.0 mg L−1 IBA.

Somatic embryogenesis is an important application of plant tissue culture for rapid and mass propagation of plants, germplasm conservation, and genetic improvement (Guan et al. 2016; Zhang et al. 2021). The green callus obtained from stem explant resulted in embryogenic callus formation whereas the brown and yellowish white callus resulted in non-embryogenic callus. Similarly, results were obtained in Camellia oleifera Abel (Zhang et al. 2021). Somatic embryogenesis is highly influenced by various parameters like the source of explant, age of culture, growth hormones, and cultural conditions (Varis et al. 2018; Hapsoro et al. 2020). In this study, the SE induction was observed on different concentrations of TDZ (0.5 and 1.0 mg L−1) and BAP (1.5, 2.0, and 2.5 mg L−1). The germination of SEs is a crucial step in the regeneration of a whole plant. In this study, germination of SEs was observed on 1.0 to 2.5 mg L−1 BAP and 0.5 to 2.0 mg L−1 NAA. In Solanum nigrum, the somatic embryogenic induction and germination were observed on 0.5 mg L−1 NAA and 1.0 to 3.0 mg L−1 BAP, and 0.5 mg L−1 BAP and 0.5 to 6.0 mg L−1 NAA, respectively (Sharada et al. 2019). In Carica papaya, 100% germination of SEs was also observed on 0.2 mg L−1 NAA and 0.2 mg L−1 BAP (Bukhori 2013). After culturing the SEs onto MS medium added with 0.5 mg L−1 BAP, the SEs developed into shoots (Ku and Chan 2013). Lema-Rumińska et al. (2019) induced SEs on BAP and NAA in Echinacea purpurea.

The histological and morphological studies also confirmed the development of SEs. In many other observations, histological, morphological, and SEM analyses were used for confirmation of SEs (Aslam et al. 2014; Shashi and Bhat 2021(Cenchrus ciliaris); Haradzi et al. 2021 (Citrus x meyeri)).

The plants originated via tissue culture may show genetic variation. This instability can be checked by various molecular assays, cytogenetic analysis, and biotechnological tools (Das et al. 2013). FCM is often used for rapid and reliable estimation of DNA content and change in ploidy levels (Bennett and Leitch 2005; Bennett and Leitch 2011). In this study’s experimentation, FCM analysis of in vivo plant, direct regenerated plant, and somatic embryogenic plant was performed for genome size analysis and genetic stability. The peaks of 2C DNA content of in vivo plant, direct regenerated plant, and somatic embryo regenerated plant showed similarity and were almost similar to in vivo plants. Thus, the DNA contents of tissue culture–raised A. maritima L. plants were unaltered. The genome size analysis and ploidy identification could be investigated by FCM for both in vivo plants and in vitro regenerated plants (Sliwinska and Thiem 2007; Sliwinska 2018). Similar genome size stability has been testified in other in vitro grown plants like Eucalyptus globules (Ribeiro et al. 2016), Camellia sinensis L. (Samarina et al. 2019), Coriander sativum (Ali et al. 2017), and strawberry cultivars (Naing et al. 2019). In certain cases, in vitro stresses brought about genome variability as was eminent in regenerated plants of Elaeis guineensis (Giorgetti et al. 2011).

Conclusions

In conclusion, this study established a novel, efficient, and genetically stable protocol for in vitro regeneration of Artemisia maritima L. via both organogenesis (direct and indirect) and somatic embryogenesis. The optimal callus induction was obtained on 2.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg L−1 6-benzylaminopurine (BAP), and shoot regeneration was observed on different concentrations of BAP, α-naphthaleneacetic acid (NAA), and thidiazuron (TDZ) using nodal segments and microshoot tips as explants. Maximum rooting was obtained on 1.5 mg L−1 NAA, and the maximum somatic embryogenic induction frequency was induced on MS medium amended with 1.0 mg L−1 TDZ and 2.5 mg L−1 indole-3-butyric acid (IBA). The establishment of somatic embryos can be utilized for the development of whole plant and true-to-types as the genetic stability was confirmed by flow cytometry. The standardized protocol would be useful in germplasm conservation and the formation of high-value clonal regenerants for commercial production. The somatic embryos could be utilized for the development of synthetic seeds and grown anywhere.