Introduction

Rheum Linn. (Polygonaceae) commonly known as rhubarb is a highly diversified genus consisting of about 60 extant species of perennial herbs. Different traditional systems of medicine are witness to the wide and efficacious use of rhubarb since antiquity to cure a wide range of ailments related to the circulatory, digestive, endocrine, respiratory, and skeletal systems. The remedying properties of Rheum are ascribed to a set of diverse bioactive secondary metabolite constituents, particularly anthraquinones and stilbenoids, besides the dietary flavonoids known for their putative health benefits (Zargar et al. 2011; Jeelani et al. 2017; Pandith et al. 2018). R. spiciforme Royle Syn. R. scaberrimum Lingelsheim ex Limpricht (web link - 2) is a perennial stemless herb with purgative root used as medicine. The species bears white green, unbranched spike like panicle (panicle spiciform) inflorescence and produces tri-winged pinkish red seeds (FOC Vol. 5 pp 349; FOC—Flora of China). R. spiciforme is a vulnerable (Kala 2005) medicinal herb that grows at an altitude of about 3047 to 4,876 m above mean sea level (asl) (Kumar et al. 2011). Besides being used as food/vegetable (leaves), the roots of R. spiciforme are also used as coloring agent by locals in the areas of its occurrence (Dorjey et al. 2012).

In comparison to the long-going conventional approaches of multiplication of a particular species, in vitro micropropagation offers a clear advantage for the propagation and multiplication at large scale (Acemi 2020). Roggemans and Claes carried out the preliminary study on in vitro propagation of the rhubarb (R. rhaponticum) and noticed initiation of the axillary buds from shoot tips grown on MS medium fortified with IBA (indole-3-butyric acid) and BAP (6-benzylaminopurine) (Roggemans and Claes 1979). This was followed by additional investigations, though inadequate, which were focused on other species of this perennial herb, and include R. palmatum (Ishimaru et al. 1990; Cui et al. 2008), R. tanguticum (Xu et al. 2004), R. ribes (Sepehr and Ghorbanli 2005), R. rhabarbarum (Lepse 2007; Rayirath et al. 2011), R. officinale (Ji-yong 2010), R. webbianum (Rashid et al. 2014), R. moorcroftianum (Maithani 2015), and R. coreanum (Mun and Mun 2016). Indeed, a detailed report on the in vitro multiplication of one of the important species of Rhubarb, R. australe, is available in our recent communication (Pandith et al. 2018). Nonetheless, and owing to the pharmacological utility of Rhubarb species, these reports do not account much toward the in vitro regeneration system of this important medicinal herb. But, as a prelude, it provides a platform for advanced empirical studies imperative for a better and valuable output.

Pandith et al. (2020) has recently reviewed the utility of the technique of in vitro multiplication of some rhubarb species. However, to date, and to the best of our knowledge, there are no authentic reports on the detailed in vitro propagation of R. spiciforme, somatic embryogenesis, in particular. Therefore, and in light of the threatened and endemic nature of R. spiciforme, the aim of present study was to develop reproducible in vitro systems to produce regenerated plants through improved seed germination, indirect organogenesis through callus, and direct somatic embryogenesis from leaf explants. We speculated that explants are affected by PGRs (plant growth regulators) and the medium composition. Therefore, the regeneration capacity of four types of explants with effect of six PGRs on seed germination, callusing, shoot multiplication, and direct somatic embryogenesis were investigated. Also, the effects of different additives like CaCl2, KNO3, glutamine and adenine sulfate were evaluated. The protocol could be utilized in large-scale propagation, germplasm conservation, genetic transformation, and industrial production of important metabolites of therapeutic significance of this important medicinal herb.

Materials and Methods

Seed collection and sterilization

The mature seeds of R. spiciforme were collected from Lachulangla, Leh, Ladakh, India (33° 04′ 53.49″ N/77° 38′ 08.32″ E; altitude 4,750 m asl) on 30th of July 2019 (Fig. 1A). The plant specimen was also deposited at the University of Kashmir Herbarium (KASH) receiving the voucher specimen number 2745-(KASH). The seeds were shade dried and stored in netted packets until use. Before transfer to culture medium, the seeds were surface sterilized according to Kauth et al. (2006) with certain modifications. Briefly, the seeds were thoroughly washed under tap water for about 2 h and treated with carbendazim 50% WP (Bavistin, Crystal Crop Protection, New Delhi, India) for 30 to 40 min to be washed again for another 30 min to remove any traces of Bavistin. Following this, they were treated with 1% (v/v) Tween 20 detergent (Sigma Aldrich, Saint Louis, MO) for 30 min and rinsed two times with distilled water. The seeds were then kept for imbibition in autoclaved water at room temperature (25 ± 2°C) in dark for 24 h. Meanwhile, all other materials required for culture were washed with laboline detergent, oven dried, and surface sterilized with 70% alcohol and autoclaved at 121°C and 15 psi pressure for 18 min. Finally, the seeds were treated with 0.1% of HgCl2 for 5 min followed by 5 to 6 rinses with autoclaved double distilled water prior to inoculation.

Figure. 1
figure 1

Seed germination and callusing in different explants of Rheum spiciforme Royle. (A) Plant in natural habitat; (B) seed germination at 0.005 mM gibberellic acid (GA3), 1 mM potassium nitrate, and 1 mM calcium chloride, (C) initiation of callusing from embryonic axis on medium with 8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 8 μM thidiazuron (TDZ); (D) regenerative brown callus on medium containing 6 μM 2,4-D and 6 μM 6-benzylaminopurine (BA); (E) non-regenerative pale callus on medium containing 4 μM 2,4-D and 4 μM BA; and (F) non-regenerative white callus on medium containing 2 μM 2,4-D and 2 μM BA.

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Seed germination

Following sterilization, the seeds were transferred to culture medium under aseptic conditions in laminar hood. The seed germination process was evaluated on Murashige and Skoog (MS; Murashige and Skoog 1962) full-strength medium (HiMedia, Mumbai, India) and MS-half strength medium supplemented with 30 g L−1 sucrose (HiMedia) and 0.8% (w/v) agar–agar (Sigma Aldrich). Both the medium types were augmented with different concentrations and combinations of gibberellic acid (GA3), calcium chloride (CaCl2), and potassium nitrate (KNO3) which were purchased from Duchefa Biochemie (Haarlem, The Netherlands). The pH of the medium was set to 5.8 ± 0.2, heated until complete dissolution, poured into glass culture vials (25 × 200 mm) and autoclaved at 121°C temperature, 15 lb pressure for 18 min. The sterilized seeds were inoculated in glass culture vials at 25 ± 2°C with 55% humidity under 12-h photoperiod (50 to 60 mol m−2 s−1) in a plant growth chamber. One seed was inoculated per vial, and 20 vials were used for each treatment. The observations were made with respect to each non-contaminated seed (experimental unit). Another set of sterilized seeds (20 seeds in each block with 5 replications) was sown in coco peat instead of the MS medium. Further, seeds (20 seeds in each block with 5 replications) were subjected to observe the germination percentage ex vitro using garden soil (soil/sand; 3:1 ratio) in pots inside greenhouse at 25°C. The seed germination was monitored on daily basis and data recorded. The mean germination time (MGT) was calculated according to Darrudi et al. (2014) using the equation:

$$ MGT=\frac{\sum \left(n\times D\right)}{N} $$

where n is the number of germinated seeds between recording intervals, D number of d from the beginning of the test, and N the total number of germinated seeds in the treatment at the end of experiment.

The percentage of seed germination was calculated using the equation:

$$ \%\mathrm{seed}\ \mathrm{germination}=\left(\mathrm{n}/\mathrm{N}\right)\times 100 $$

where n is the total number of germinated seeds and N the number of seeds used at the beginning of the experiment.

Callusing

For callogenesis, node, leaf, root, and embryo segments were used as explants. Node, leaf, and root segments of about 1 cm were excised from the aseptically grown 6-wk-old in vitro seedlings, 4.9 ± 0.2 cm in height. In case of embryo, the surface sterilized seeds described in the previous section were used. As the seeds were swollen by imbibition, the embryos were excised with the help of scalpel and cut at both sides (from radicle and cotyledon side) to use the main embryonic axis (0.3 cm in length) for callusing. The segments of node, leaf, and root and embryonic axis were cultured in glass culture vials with 20 vials per treatment in MS medium supplemented with 0.0 to 10.00 μM 2,4-D (2,4-dichlorophenoxyacetic acid), BAP, KIN (kinetin) and TDZ (thidiazuron) in all suitable combinations for callogenesis. The callus derived after 7 wk was transferred on medium augmented with 2.0 to 10.00 μM BAP, KIN, and GA3 for organogenesis. These additives were procured from Duchefa Biochemie. The percentage regeneration was calculated with respect to callus derived from different explant sources. Here, the regenerative callus is defined as the callus found capable of developing somatic embryos. The parameters like color, response percentage, and growth were evaluated. Moreover, response percentage of explants was calculated by counting the number of explants responded divided by total explants used and multiplied by 100. Furthermore, the growth of callus was evaluated in terms of volume (cm3) of callus formed after 36 d of inoculation. The growth was expressed as –, +, ++, +++, and ++++ which indicates no, slight (less than 2 cm3), moderate (2 to 4 cm3), intense (4 to 6 cm3), and very intense (more than 6 cm3) callogenesis (growth), respectively.

Somatic embryogenesis

For somatic embryogenesis, like callusing, single intact axillary leaves were used from the aseptically grown 6-wk-old in vitro seedlings, 4.9 ± 0.2 cm in height. The leaves along with petioles in both upright and horizontal (keeping abaxial side up in some explants and adaxial side in others) positions were cultured in glass culture vials with 20 vials per treatment containing MS medium fortified with 5.0 to 30.00 μM BAP, 0.0 to 01.00 μM NAA (naphthaleneacetic acid), 2.0 to 8.00 μM 2,4-D, 2.0 to 8.00 μM GA3, 25.0 to 100.00 μM GTM (glutamine), and 25.0 to 100.00 μM ADS (adenine sulfate) in all suitable combinations. The optimal condition for direct somatic embryogenesis in culture media at 25 ± 2°C with 55% humidity in dark for 1 wk was achieved by keeping vials inside a container covered completely by aluminum foil inside a plant growth chamber. Parameters including location of somatic embryo formation, number of somatic embryos formed, and percentage regeneration of these somatic embryos into plantlets were evaluated against each treatment.

Multiple shoot induction

Nodal segments were excised from the aseptically grown 6-wk-old in vitro seedlings, 4.8 ± 0.5 cm in height. The single segments of about 1 cm were cultured in glass culture vials with 20 vials per treatment in MS medium supplemented with 0.0 to 30.00 μM BAP, 0.0 to 01.00 μM NAA, 1.0 to 15.00 μM KIN, 5.0 to 100.00 μM GTM, and 1.0 to 100.00 μM ADS in all possible combinations for shoot multiplication and evaluation of the effects of different combinations on shoot morphology. The in vitro regenerated shoots were multiplied and maintained by repeated transfer of mother explants and sub-culturing of in vitro raised shoots and callus. The mean number of shoots per explant, mean length of shoot, color of shoots and vigor of lamina, base callusing, percentage response and relative growth of callus in volume, location, number, and percentage regeneration of somatic embryos were recorded. Moreover, for shoot multiplication, callusing, and induction of somatic embryogenesis, the culture medium was initially supplemented with basic PGRs, and after recording the first set of results, the concentration of these PGRs was kept constant to evaluate the effect of others, and the trend was followed in each case.

Adventitious root induction and acclimatization

The plantlets generated following the above-mentioned procedures were transferred for rooting to culture vials containing half-liquid Murashige and Skoog (LMS) medium and filter-bridge augmented with IAA (indole-3-acetic acid), IBA, and NAA at 0.5 to 2.5 μM concentrations. The one-half LMS medium devoid of any PGR was taken as control. Rooted seedlings from the culture vials were removed and transferred to thermocol cups having sanitized Soilrite™ (75% peat moss and 25% horticulture mark expanded perlite) (Keltech Energies Ltd., Bangalore, India). During hardening the plants were also sprinkled with quarter-strength MS inorganic solution and covered with polyethylene bags. The covered plantlets were kept in culture room at 25°C, and polyethylene bags were removed after 2 wk. The healthy plants were hardened finally in garden soil after keeping the pots in greenhouse for 4 wk and relocated to natural conditions subsequently.

Statistical analyses

Each experiment was replicated three times with 20 samples in each, and the results were recorded after 6 wk. The results are expressed as the mean ± standard error. The treatments presented and compared in each table were evaluated in experiments carried out at same time, and the statistical analyses were carried out separately for each experiment. The data collected was analyzed in SPSS software (Version 25, SPSS Inc. Chicago, IL). The significance, analysis of variance, and significant differences among the treatments were determined based on one-way ANOVA and assessed following Tukey’s and LSD Posthoc tests at 95% confidence limits (P < 0.05).

Results

Seeds exhibited better germination on half-strength MS medium supplemented with gibberellic acid, calcium chloride, and potassium nitrate

Seeds of R. spiciforme showed a fairly significant increase in volume within the first 24 h when imbibed at room temperature (25 ± 2°C) in the dark. The percentage of seed germination in R. spiciforme was better in half-strength MS medium in comparison to full-strength MS medium. In MS-full medium, the percentage seed germination was the highest (71.5 ± 1.4%) with mean germination time (MGT) of 14.0 ± 1.7 d when supplemented with 0.005 mM GA3, 1 mM CaCl2, and KNO3 each. On the other hand, in half-strength MS medium, the addition of 0.005 mM GA3 significantly increased the rate of germination from 25.0 ± 0.6 to 75.2 ± 1.2 % with MGT of 10.6 ± 2.0 d (Table 1). So, while keeping the concentration of GA3 constant at 0.005 mM, addition of 1.0 mM CaCl2 was seen to increase the rate of seed germination to 91.4 ± 2.2 % and lowered the MGT to 9.1 ± 1.7 d. Moreover, the seed germination increased to 92.6 ± 1.3 % when 1 mM KNO3 is supplemented along with 0.005 mM GA3 and 1 mM CaCl2 with MGT of 8.5 ± 1.8 d (Fig. 1B). The seeds were also grown in garden soil and coco-peat, and percentage germination of seeds in soil is very low (13.6 ± 3.1) compared to the coco-peat (58.5 ± 2.3 %) (Table 1).

Table 1 Effect of gibberellic acid (GA3), calcium chloride (CaCl2), and potassium nitrate (KNO3) fortified Murashige and Skoog (MS) medium on seed germination of Rheum spiciforme Royle.
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Callogenesis: 2,4-dichlorophenoxyacetic acid and 6-benzyl amino purine worked better on leaf and nodal explants; thidiazuron exhibited profound effect on the embryo

In this study, four types of calluses were found in different explants with respect to different concentration and combination of PGRs (Table 2). Nevertheless, embryos responded with 67.9 ± 1.8% non-conspicuous callusing on medium containing 8 μM 2,4-D and 8 μM TDZ (Fig. 1C). The brown callus (Fig. 1D) was observed to undergo organogenesis. On the other hand, pale yellow- (Fig. 1E) and white- (Fig. 1F) colored callus cultures were very compact and feathery in appearance as compared to the loose and soft brown calluses. The red-colored callus was also found to be non-viable. These observations can be correlated to Table 2. At lower concentrations of 2,4-D and BA (below 2 μM), white callus was formed in leaf and nodal explants with moderate growth. The percent callus response from leaf, node, and root explants increased with addition of 2,4 D and BA to the medium. Among four types of explants used for callusing, 87.4 ± 1.5% leaf explants responded at 2 μM 2,4-D and BA each; at 4 μM 2,4-D and BA, the percent response of explants decreased (56.2 ± 1.9%) with increased callogenesis. The callogenesis in node and root was best observed at 4 μM (81.7 ± 1.2%) and 6 μM (79.8 ± 1.6%) 2,4-D and BA each, respectively (Table 2). Although KIN in leaf and nodal explants slightly improved the percent response, however, callus died in third wk of culture. When KIN was supplemented along with 2,4-D and BA, it improved the formation of regenerating brown callus. The application of TDZ however brought no significant effect on leaf, nodal, and root explants in this study. Pertinently, in embryo, TDZ was found to increase the percent response of explants along with the rate of callogenesis. Nevertheless, the callusing was not prominent in case of embryo explants.

Table 2 Effect of different plant growth regulators (PGRs) on callusing from leaf, node, root, and embryo explants of Rheum spiciforme Royle.
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Morphogenesis of regenerative callus: nodal segments displayed vibrant regeneration with intense rooting that shifted toward shooting with gibberellic acid

The ultimate aim of callusing is to produce shoots for speedy propagation. In this study, callus transferred on medium fortified with equal concentration of BA and KIN only started the formation of shoot buds on leaf explant-derived callus (Fig. 2). Regeneration percentage of callus also varied with respect to explant used. The highest regeneration percentage of 81.3 ± 0.9 was observed in nodal segment-derived callus followed by calluses from leaf, embryo, and root with intense rooting at 8.0 μM BA and KIN each (Table 3). However, direct rooting from calluses was also observed which increased with increase in concentration of BA and KIN (Fig. 2A). To overcome this problem, GA3 was supplemented along with BA and KIN. Addition of GA3 improved bud maturation and regeneration percentage and decreased the emergence of rootlets (Fig. 2B–D). The highest percentage of regeneration (96.3 ± 1.6%) was observed in brown calluses derived from nodal segments followed by leaf (87.2 ± 1.5%) at 8.0 μM BA, KIN, and 4.0 μM GA3 with slight rooting. In calluses derived from embryonic axis, lower (2 μM) concentration of GA3 was found effective with percentage regeneration of 74.3 ± 0.7%. It was found that root-derived callus responded least to any supplement combination with highest regeneration rate of 43.2 ± 2.1% at 8.0 μM BA and KIN each and 6.0 μM GA3 (Table 3).

Figure. 2
figure 2

Regeneration and organogenesis in callus from Rheum spiciforme Royle. (A) Root initiation on leaf derived callus at 8 μM 6-benzylaminopurine (BA) and 8 μM kinetin (KIN); (B) initiation of shoot bud formation in node-derived callus at 8 μM BA, 8 μM KIN, and 4 μM gibberellic acid (GA3); (C) fully differentiated shoot from node-derived callus at 8 μM BA, 8 μM KIN, and 6 μM GA3; and (D) regenerated plantlet from node-derived callus at 8 μM BA, 8 μM KIN, and 6 μM GA3.

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Table 3 Effect of different plant growth regulators (PGRs) on percentage regeneration of callus derived from leaf, node, root, and embryo explants of Rheum spiciforme Royle.
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Axillary leaves lead to the direct induction of regenerative somatic embryos from both abaxial and adaxial sides of lamina and petiole

The present investigation showed that BA at 15 to 25 μM concentration along with 1.0 μM NAA (Table 4) induces the formation of somatic embryos directly from leaf explants by culturing single intact axillary leaves on MS full medium. Formation of somatic embryos on adaxial side was observed after 2 wk. Initially, the leaf explants developed globular structures on adaxial side which later on grew into dark green shoots. When the concentration of BA was increased to 25 μM, emergence of mean 7.8 ± 0.9 somatic embryos were observed at juncture of lamina and petiole on adaxial side (jads) (Fig. 3A–B), juncture of lamina and petiole on abaxial side (jabs) (Fig. 3C), lamina on adaxial side (lad), and petiole (attached to lamina) (Fig. 3D) with 48.3 ± 1.7% regeneration. To increase the regeneration of somatic embryos into complete plantlets (Fig. 3E–F), along with 25 μM BA and 1.0 μM NAA, 2,4-D, GA3, GTM and ADS were also added to the medium. Indeed, the addition 4 μM 2,4-D improved the rate of germination of somatic embryos up to 62.9 ± 2.9% which was further increased to 76.1 ± 2.3% by adding GA3 at a concentration of 8 μM. It was observed that although mean number of somatic embryos per explant decreased to 6.2 ± 0.9, the percentage regeneration increased to the maximum of 91.0 ± 3.5% at 25 μM BA, 1.0 μM NAA, 2.0 μM GA3, and 50.0 μM GTM and ADS each. Also, it was found that maximum of 3.9 ± 1.0 plantlets were obtained from each explant fortified with 25 μM BA, 1.0 μM NAA, 2.0 μM GA3, and 50.0 μM GTM and ADS each (Table 4).

Table 4 Effect of different plant growth regulators (PGRs) on direct somatic embryogenesis from leaf explants of Rheum spiciforme Royle.
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Figure. 3
figure 3

Direct somatic embryogenesis in Rheum spiciforme Royle. (A–B) Somatic embryos formed on adaxial side of lamina at 25 μM 6-benzylaminopurine (BA) and 1 μM naphthaleneacetic acid (NAA); (C) somatic embryos formed on abaxial side of lamina at 25 μM BA and 1 μM NAA; (D) somatic embryos formed on petiole at 25 μM BA and 1 μM NAA; (E) fully grown somatic embryo at 25 μM BA, 1 μM NAA, 4 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and 2 μM gibberellic acid (GA3); and (F) regeneration of shoots from somatic embryos at 25 μM BA, 1 μM NAA, 2 μM GA3, 50 μM glutamine (GTM), and 50 μM adenine sulfate (ADS).

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A nitrogenous base (adenine sulfate) and an amino acid (glutamine) exhibited combinatorial effect in inducing extensive shoot multiplication in nodal segment-raised plants

When single axillary shoots were grown on culture medium supplemented with 12.5 μM BA and 0.5 μM NAA, these multiplied extensively and produced mean 27.2 ± 2.9 shoots with mean shoot height of 3.4 ± 0.6 cm (Table 5). Nonetheless, it was observed that although shoot multiplication took place, shoots were found with underdeveloped lamina and pale-colored petioles (Fig. 4A). On the other hand, GTM and ADS supplemented at 25 μM concentration each (and not alone) along with 12.5 μM BA and 0.5 μM NAA produced mean 10.2 ± 2.6 shoots with mean 4.2 ± 0.4 cm shoot height, well developed green lamina with better overall vigor (Fig. 4B–D; Table 5). Therefore, for extensive shoot multiplication and maintenance of overall vigor, we suggest to grow single axillary explants on 12.5 μM BA and 0.5 μM NAA for 3 wk for multiplication and then transfer the culture on medium supplemented with 25 μM GTM and ADS each along with 12.5 μM BA and 0.5 μM NAA for 7 wk.

Table 5 Effect of different plant growth regulators (PGRs) on shoot multiplication, mean height of shoots, and overall vigor of Rheum spiciforme Royle.
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Figure. 4
figure 4

Shoot multiplication, rooting, and acclimatization of Rheum spiciforme Royle. (A) Shoot multiplication (pale yellow) at 12.5 μM 6-benzylaminopurine (BA) and 0.5 μM naphthaleneacetic acid (NAA); (B) multiple shoot formation at 12.5 μM BA and 0.1 μM NAA; (C) shoot elongation and multiplication at 12.5 μM BA, 0.5 μM NAA, and 25 μM glutamine (GTM); (D) shoot elongation and multiplication at 12.5 μM BA, 0.5 μM NAA, 25 μM GTM, and 25 μM adenine sulfate (ADS); (E) rooting at 2.5 μM NAA; and (F) hardening.

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Naphthaleneacetic acid induced adventitious root system required for in vitro plants’ acclimatization under natural conditions

The plantlets initiated rooting in half-LMS supplemented with 2.5 μM NAA after 3 wk (Fig. 4E). The rooted plantlets were transferred to thermocol cups filled with sanitized Soilrite and hardened for 3 wk and subsequently transferred to pots in greenhouse (Fig. 4F).

Discussion

In vitro micropropagation/regeneration technique has the potential to provide an organized production system with uniform quality and yield. It has proven to be a useful asset for easy production of similar individuals of a particular genotype (Pandith et al. 2018). Tissue culture has been extensively employed for the in vitro propagation, regeneration, production of pharmaceutically important bioactive compounds, and germplasm conservation of rare and endangered medicinal herbs (Das and Rout 2002). Therefore, the present study was focused on production of the reproducible in vitro regeneration and multiplication system of an endemic and vulnerable medicinal herb R. spiciforme.

The seeds of Rheum have been shown to bear moderate levels of seed dormancy (Rutherford and Ali 1977) imposed by embryo and certain inherent inhibitors (Farzami and Ghorbanli 2011). Seed germination in Rheum is known to be species specific as well as habitat specific and exhibits susceptibility to even moderate changes in soil water condition (Bo Song 2013). In the present study, seed germination improved to 92.6 ± 1.3% on half-strength MS medium fortified with 1 mM KNO3, 0.005 mM GA3, and 1mM CaCl2 with MGT of 8.5 ± 1.8 d. Similar results have been found in some other species of Rheum viz. R. khorasanicum (Darrudi et al. 2014). The GA3 promotes seed germination by stimulating the growth potential of embryo and release of hydrolases that weaken the structures surrounding embryo (Kamiya et al. 2002). On the other hand, calcium improves seed germination by loosening the cell wall expanding highly hydrated gel networks (Lapasin and Pricl 1995), by providing structural integrity to cellular membranes (Burstrom 1968), and by regulating the activity of certain kinase and phosphatase enzymes that play important role in the signal transduction during seed germination process (Derek 1997; Harper et al. 2004). Although KNO3 and other nitrogenous compounds elevate levels of seed viability and germination in many crop and tree species (Beligni and Lamattina 2000; Bethke et al. 2004; Gniazdowska et al. 2007; Gao et al. 2011; Gupta et al. 2011), the combined effect of CaCl2 and KNO3 has been found to be more pronounced than KNO3 alone in R. khorasanicum (Darrudi et al. 2014). The nitrates possibly remove seed dormancy through pentose phosphate pathway and consume oxygen evolved during various oxidation processes during seed germination (Finch-Savage and Leubner-Metzger 2006). Moreover, KNO3 and CaCl2 are also known to break dormancy and promote germination in R. ribes (Farzami and Ghorbanli 2011).

The induction and initiation of callusing is regulated by the relationship between cytokinin and auxin levels in the plant, with their contents being species and genotype specific (Gutiérrez et al. 2011). Auxins and cytokinins have been observed to show remarkable effects on growth, differentiation, and metabolism of cultured cells (Duangporn and Siripong 2009). Among auxins, 2,4-D is considered the best for initiation of callogenesis and is widely used in both monocot and eudicot callus cultures (Evans et al. 1981; Ho and Vasil 1983; Chee 1990; Mamun et al. 1996). The callus induction in most of the plant species follows classical equal cytokinin to auxin ratio (Skoog and Miller 1957). BA and KIN along with 2,4-D have been commonly used for callus induction (Chai and Sticklen 1998). In the present study, nodal and root explants responded best at 4 μM (81.7 ± 1.2) and 6 μM (79.8 ± 1.6%) 2,4-D and BA. Comparatively, in R. moorcroftianum, calluses were observed on MS basal medium containing 6.5 μM BA and 11.5 μM IAA (Maithani 2015). The formation and regeneration of different types of callus as a consequence of varying combination and concentration of PGRs is also observed in other species like Coryphantha elephantidens (Wakhlu and Bhau 2000), Nerium odorum (Rashmi and Trivedi 2014), and Tussilago farfara (Ren et al. 2017). Interestingly, in R. spiciforme embryo, TDZ was found to increase the percent response of embryo explants (67.9 ± 1.8%) along with very intense callusing at 8 μM 2,4-D and TDZ each. Indeed, TDZ, a synthetic phenylurea derivative, has shown to possess activities higher than adenine derivatives (Mok et al. 1982).

In different medicinal plants, like Oroxylum indicum, the two cytokinins BA and KIN have been used for in vitro regeneration and organogenesis where BA exhibited high percentage of shoot buds as compared to the KIN (Gokhale and Bansal 2009). BA is an important cytokinin and plays a key role in differentiation and formation of adventitious shoots (Fang et al. 2018; Tan et al. 2018). The efficiently metabolized phytohormone has been shown to stimulate the production of natural hormones in plant tissues leading to induction of organogenesis (Abbasi et al. 2013). The highest regeneration percentage observed in nodal segments with a proportionate percentage in the same explant derived brown calluses corroborates with similar results reported in Oryza sativa (Ray et al. 1996), Arabidopsis thaliana (Ikeda-Iwai et al. 2002), Coffea arabica (Quiroz-Figueroa et al. 2002), and Triticum aestivum (Munazir et al. 2010). The callus in most of the in vitro experiments consists of different cell types, and only some of them could be involved in organ regeneration (Feher 2019) as seen in tobacco inter-nodal explants where shoot regeneration competence was diminished in more mature or elongated cells (Gilissen et al. 1996). This might be the reason that out of four types of calluses formed, only brown callus was found regenerative. In studies with similar results, GA3 has been shown to induce very positive effect on regeneration responses in otherwise very poor responsive calluses and improved induction and germination of shoot buds (Hunault and Maatar 1995; Viéitez Martín and Barciela 1990).

The process of direct somatic embryogenesis has been used in several medicinal plants such as Gymnema sylvestre (Kumar et al. 2002), Tylophora indica (Jayanthi and Mandal 2001), Psoralea corylifolia (Chand and Sahrawat 2002), Gymnema sylvestre (Kumar et al. 2002), and Holostemma ada-kodien (Martin 2003). Importantly, in genus Rheum, R. emodi has been shown to produce direct shoot buds from leaves in medium containing 10.0 mM BA and 5.0 mM IBA (Lal and Ahuja 1989). However, in this study, it was observed that higher levels of BA (25 μM) along with 1.0 μM NAA induce direct somatic embryos in R. spiciforme. These results were in consonance with other studies on Fragaria × ananassa (strawberry), Polygonum rosea, and P. zeylanica in the medium containing BA in combination with IBA (Liu and Sanford 1988; Das and Rout 2002). Cell clusters formed in the cultures led to the generation of somatic embryos. This clustering and isolation of cells has been shown to lead to reprogramming of genomic and cellular functions essential for the acquisition of embryogenic competence (Verdeil et al. 2001). Moreover, the final response following use of PGRs may be associated with two or more hormones or related additives wherein one may induce the synthesis of another (Taiz and Zeiger 1991). The low regeneration issue of the direct somatic embryos was resolved by addition of GA3, GTM, and ADS as later two have been shown to improve the induction and regeneration of somatic embryos in other species like P. rosea and P. zeylanica (Das and Rout 2002; Ipekci and Gozukirmizi 2004). Additionally, one of the main nutrients in plant tissue culture media is nitrogen with which cytokinins interact. A positive interaction of cytokinins in the regulation of enzymes is associated with the assimilation of this element which leads to the activation of the nitrate reductase enzyme thereby promoting cell multiplication (Samuelson et al. 1995).

Extensive shoot multiplication is the ultimate aim of tissue culture technique. Shoots derived either from germination of seeds, organogenesis in vitro, or direct somatic embryogenesis need to be multiplied for either transplantation into natural conditions or to extract constituents of therapeutic significance (in case of medicinal plants). Although the contents of culture media involve various essential minerals, each plant species has its specific elemental requirements and responds differently to various additives and media formulations (George et al. 2008). In rhubarbs, like R. rhaponticum, shoot multiplication has been shown to take place on medium supplemented with different concentrations of BA and IBA (Roggemans and Claes 1979; Lal and Ahuja 1989; Lassus and Voipio 1994). BA plays a key role in the process of induction of shoots in R. rhaponticum and in other Polygonaceae members including Coccoloba uvifera (Wojtania and Gabryszewska 2000), Rumex acetosella, R. acetosa (Ćulafić et al. 1987), and Polygonum aubertii (Dabski and Kozak 1998). In the current study, 12.5 μM BA and 0.5 μM NAA in addition to 25 μM GTM and ADS were found optimal for shoot multiplication. Interestingly, our results are consistent with the concept that higher concentrations of cytokinin in combination with lower auxin concentration in the shoot induction medium favor shoot formation (Sugimoto et al. 2011). Moreover, KIN is also known to play a role in shoot multiplication; however, no significant improvement was found in this study in comparison to other studies on Leptadenia reticulata (Shekhawat et al. 2006). Pertinently, studies on Sarcostemma brevistigma and Sapindus trifoliatus have advocated the superiority of BA over KIN in shoot induction as well as its multiplication (Thomas and Shankar 2009; Bisht et al. 2012a, b) probably due to the ability of plant tissue to metabolize BA more readily as compared to other cytokinins used for shoot multiplication (Abbasi et al. 2013). Certainly, to improve the overall vigor, adenine supplemented in various forms, including ADS, is known to induce the proliferation of axillary shoots and promote the adventitious shoot formation in different types of explants (Van Stedan et al. 2008). Additionally, the effectiveness of organic nitrogen source for shoot multiplication in the form of GTM has also been reported by many authors (Green et al. 1990; Vasudevan et al. 2004). Indeed, in order to improve the process of shoot multiplication and maintain the overall vigor, ADS supplied in combination with GTM has been proven more effective (Siwach and Gill 2011).

According to Mosca et al. (2017) and Teardo et al. (2019), it is the osmotic aspect of medium which affects root formation and development, and exogenous PGRs do regulate the osmotic pressure. Also, NAA, a synthetic auxin, has been shown to play a crucial role during root formation and development (Martinez-de la Cruz et al. 2015; Cui et al. 2019). In this study, however, the success rate was recorded below 33%. There are limited reports on the acclimatization of rhubarbs’ obtained from in vitro cultures (Lal and Ahuja 1989). The probable reasons are that rhubarb plants are very difficult to be stabilized phenotypically after in vitro treatments (Zhao et al. 2002). Moreover, according to Zhao et al. (2006), the application of PGRs and their different concentrations cause morphological deformations in rhubarb plants which result in their low resistance. The changes may be a result of environmental factors and an inappropriate selection of a given genotype for in vitro propagation (Zhao et al. 2005, 2007).

Conclusions and Outlook

In conclusion, owing to the restricted zone of R. spiciforme besides the natural and anthropogenic interventions which threaten its survival, the plant species deserve immediate and effective attention to formulate sustainable conservation measures. In this context, the present study describes an efficient and reproducible in vitro regeneration and multiplication protocol for the large-scale propagation of R. spiciforme utilizing three different pathways: organogenesis, embryogenesis, and shoot proliferation. This study showed that the processes of seed germination, callus regeneration, shoot multiplication, and induction of direct somatic embryogenesis are affected by PGRs and other additives like adenine sulphate and glutamine. It was found that for extensive shoot multiplication and maintenance of overall vigor in this species, single axillary explants need to be cultured on medium containing 12.5 μM BA and 0.5 μM NAA for 3 wk for multiplication and then transferred on medium supplemented with 25 μM GTM and ADS each along with 12.5 μM BA and 0.5 μM NAA for 7 wk. The developed protocol with a sufficiently high success rate offers to be the starting point for development of other contemporary biotechnological avenues toward the conservation and resource management of this valued herb. In addition, this study can also be used in industrial production of biologically active secondary metabolites from in vitro derived tissues under controlled conditions for their commercial exploitation. In this study, although a complete in vitro regeneration system was established, further research is required to utilize the platform as a prelude to enhance the production of pharmaceutically important metabolites through genetic transformation techniques and bioreactor systems.