Micropropagation studies in Bergenia ligulata: A potential plant remedy to kidney stones

doi:10.21203/rs.3.rs-2320653/v1

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Micropropagation studies in Bergenia ligulata: A potential plant remedy to kidney stones

https://doi.org/10.21203/rs.3.rs-2320653/v1

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Rhizomes of Bergenia are frequently used as antiurolithiatic agents in hilly regions of India, leading to this plant becoming threatened. Furthermore, Bergenia also serves as a diuretic, antitussive, and antipyretic, and is used as a treatment for diarrhoea, vomiting, fever, cough, pulmonary infections, menorrhagia, excessive uterine haemorrhage, and stomach ulcers. Using leaf/petiole explants from Bergenia ligulata, one-step regeneration was demonstrated. MS media supplemented with 0.2 and 0.4 mg L^-1 BAP with 0.1 mg L^-1 NAA resulted in the highest number of shoots on either explant (leaf/petiole). However, multiple shoots were produced on MS media fortified with 2 mg L^-1 BAP and 0.5 mg L^-1 Kn. An efficient and rapid regeneration protocol for this plant is presented in this study.

Bergenia ligulata

Micropropagation

SEM

Direct and Indirect Regeneration

An efficient, cost-effective, and one-step in vitro regeneration protocol was reported (i.e., 3597 plants were regenerated from a single explant over a one-year period), which included molecular diversity and fidelity and SEM studies to validate direct regeneration.

Often used for kidney stones, the Bergenia plant is popularly known for its anti-uriolithiatic activity, which is attributed to its high content of bergenin in its roots. Higher altitudes in the Himalayas (1000–3500 m) are more likely to host this plant. Furthermore, it can be found in East Asia (China, Tibet), Northern South Asia (across the Himalayas in India, Bhutan, and Nepal), and South-West Asia (Afghanistan, Pakistan). It is classified as a perennial herbaceous plant and belongs to the Saxifragaceae family, which consists of 36 genera and more than 600 species.

The genus Bergenia is comprised of 30 species found across the world to date. The secondary metabolites of almost all the species of Bergenia have high pharmacological values that range from alkaloids, terpenoids, and phenols, which contribute not only as medicines but also as food and cosmetics. In India, there are three Bergenia species found: B. ligulata, B. ciliata, and B. stracheyi. Moreover, the Bergenia plant has several medicinal properties and is used as a diuretic, antitussive, and antipyretic, and used in diarrhea, vomiting, fever, cough, pulmonary infections, menorrhagia, excessive uterine hemorrhage, and ulcer of large intestines.

The possible decline in genetic diversity of medicinal plants has been noted due to indiscriminate usage, destruction, and overharvesting, which has placed these plants on the list of endangered species. Furthermore, the sharp rise in exports of medicinal and aromatic plants over the past few decades is evidence of the rising demand for these plant products on a global scale. Tissue culture, as previously noted, has the potential to be a popular alternative biotechnological tool that allows plants to flourish in a hassle-free setting. Additionally, plant regeneration through tissue culture can assist in obtaining germplasm and biomass usage for global therapeutic reasons. Few Bergenia propagation reports mention callus-mediated indirect regeneration (Rafi et al., 2016; Haq et al., 2019). The current manuscript describes the direct regeneration of Bergenia ligulata using its leaf and petiole explants, along with the protocol for inducing multiple shoots at once.

Bergenia ligulata plants were obtained in the glasshouse at CSIR-CIMAP Lucknow campus after being taken from the institute's Bhagheshwar facility in Purara, Uttarakhand. The plants were raised in clay pots using garden soil. The in-vitro cultures of Bergenia plants were started using the leaves, budding buds, and petioles.

2.2 Surface Sterilization And Culture Conditions

Young leaves, petioles, and buds were chopped and thoroughly washed under running water for surface sterilisation. Following thorough washing with running tap water, leaves were submerged in a 0.01% diluted detergent solution (labolene) to remove all traces of grime and dust. To get rid of the endophytic fungus, leaf/petiole/bud explants were also extensively cleaned with tap water 2–3 times before being thoroughly treated with Bavistin (50% W.P Systematic Fungicide) for about 5 minutes. Then, explants were surface sterilised for 8 minutes with a 0.1% (w/v) aqueous mercuric chloride solution. Explants were cleaned thoroughly with sterile, double-distilled water several times under laminar air. A 3% sucrose addition and 3 g L^− 1 Gelrite solidification were made to the MS medium. Before autoclaving, the pH was adjusted to be between 5.7 to 5.8. A cotton plug was used to seal the 100 mL conical flask after filling it with 50 mL of the MS medium with the desired growth regulator and autoclaving it at 121°C for 20 minutes.

2.3 In vitro shoot proliferation and multiplication

For direct and indirect shoot regeneration, surface sterilized leaf/petiole/axillary bud explants were inoculated on a half-strength MS medium (Murashige & Skoog, 1962) fortified with various combinations and concentrations of growth regulators like BAP with IAA, IBA, and NAA. Following the successful development of the shoots, the medium (MS) containing BA, Kn, GA3, IAA, and NAA was further transplanted for the induction of multiple shoots. The quality of shoots and the rate of culture multiplication were consistently observed at intervals every two weeks to determine the ideal growth hormone combination.

2.4 Microscopy (Sem Studies)

Regenerated plantlets were thoroughly cleaned with deionized water three times to remove any signs of agar/gel rite before being examined under a scanning electron microscope to observe the regeneration. Unprocessed samples had been mounted on aluminium stubs with copper tape that had two sticky sides, and they were then put in the SEM specimen chamber (FEI-Quanta 250). An image of the samples was captured using a large field detector (LFD) in low vacuum mode, and a chamber pressure of 120 Pa. Images were captured at a working distance of 13.3 mm and an accelerated voltage range of 15 Kv to 20 Kv. The image was obtained with a relatively modest magnification (122 X), and the ideal spot size (4.0) was selected to improve resolution.

Molecular Analysis

2.5 Extraction and quantification of Genomic DNA

Bergenia ligulata plantlets that had been in vitro micro propagated, were randomly chosen (5 plants total: C1-C5), and their corresponding mother plants' genomic DNA was extracted using a commercially available kit (DNeasy Plant Mini Kit, Qiagen, Valencia, CA, USA). The kit combines micro spin technology with the binding capabilities of silica gel-based membranes. As the concentration of chaotropic salt increases and the DNA molecules are adsorbed to the silica gel membrane, the removal of carbs, polyphenols, and other metabolites is optimized. For their qualitative and quantitative measurements, respectively, gel electrophoresis was done using the genomic DNA on a 0.8% agarose gel along with spectrophotometer analysis using a nanodrop (ND 1000, Thermo Scientific, USA).

2.6 Amplification Through Pcr

In the beginning, 10 of the 15 ISSR primers (Sigma Aldrich Chemical Pvt. Ltd., India) that were examined responded to the genetic fidelity study in the Bergenia ligulata plant (Table 3). In order to amplify inter simple sequence repeats (ISSR), a reaction mixture containing 25–30 ng of template DNA, 2 l µl of 10 X PCR buffer, 2 l µl of MgCl2 (50 mM), 1µl of dNTPs (10 mM), and 0.6 U of DNA Taq DNA polymerase (Merck Specialities Pvt. Ltd., India) was used. A thermal cycler (ProflexTM Base, Thermo Fisher Scientific, India) was used to perform the PCR amplification under the following conditions: initial denaturation at 94°C for 5 minutes, 37 cycles at 94°C for 1 minute, 53°C for 43 seconds, 72°C for 2 minutes, and final extension at 72°C for 7 minutes.

2.7 Pcr Product Validation

The acquired PCR products underwent electrophoresis on 1.2% w/v agarose gel in 1X TAE (Tris-Acetic acid-EDTA) buffer. The amplified fragments were observed using a Gel Documentation System (XR-Quantity One, Bio-Rad Laboratories, USA) under UV light. Comparisons were made between the amplified product sizes and a low-range DNA ruler plus 100–3000 bp (GeNei Pvt. Ltd, India). Only the different bands were considered for scoring, and each reaction had its replicates (Fig. 2)

2.8 Statistical Analysis

Each experiment has analyzed with 3 replicates, and the results were demonstrated in terms of mean ± standard deviation. Collected data were statistically analyzed) with a t-test by using Excel (Microsoft Office Home 2019, Version 2206) with various levels of significance (P = < 0.001, P = < 0.01, P = < 0.05).

3.1 In-Vitro shoot (direct and indirect) regeneration protocol

On MS basal media, the potential for regeneration of Bergenia ligulata leaf, petiole, and axillary bud was evaluated (half and full strength). The axillary bud failed to regenerate, although the leaves and petiole showed both direct and indirect regeneration with remarkably similar outcomes. Only leaf explants were presented for this manuscript because each of these explants showed nearly identical findings. The direct and indirect regeneration of B. ligulata leaves/petioles was examined in the present study using a variety of growth regulator combinations with MS media.

While certain hormone combinations produced callus indirectly from the explants, others produced direct shoot regeneration. Several cytokinins and auxin combinations were tested for direct shoot proliferation from leaf and petiole explants. We discovered that 1.5 mgL^− 1 BAP + 1 mgL^− 1 IBA showed 24 shoots per explant with a 51% response (Table 1, Supplementary Table 1). For direct shoot rejuvenation, BAP (0.5, 1.0, 1.5, 2.0, and 2.5 mgL^− 1) in combination with IAA (1 mgL^− 1) or NAA (0.1 or 0.5 mgL^− 1) was also examined. The combination that produced the most direct shoot proliferation (17.3 shoots per explant) and 80% response in as little as 25 days was discovered to contain 0.5 mgL^− 1 BAP and 1 mgL^− 1 IAA in the MS medium (Table 1). Additionally, the combination of 0.2 or 0.4 mgL^− 1 BAP with 0.1 mgL^− 1 NAA was discovered to be the best among the growth regulators evaluated for direct shoot initiation response, with 25.3 and 28.7 shoots/explant, respectively, and more than 95% response in 3 weeks. However, even with a lower percent response, shoot numbers were dramatically reduced by raising the concentration of NAA by 0.5 mgL^− 1 with 0.5 and 1 mgL^− 1 BAP (10 and 7.3 shoots, respectively) (Table 1, supplementary table 1).

Table 1

Effect of combinations of growth hormones on regeneration potential of wild-grown *Bergenia ligulata*
S.N.	Medium	MS medium supplemented with (mgL^− 1)						Nature of Response		Percent Response (%)	Days required for shoot initiation
		BAP	IAA	IBA	NAA	Kn	2,4-D	Direct (No of shoots)	Indirect
1.	Control	-	-	-	-	-	-	-	-	-	-
2.	MS (Full strength)	0.5	-	1.0	-	-	-	3^NS ± 1		29.3 ± 3.5	30
3.		1.0	-	1.0	-	-	-	7^NS ± 1		11 ± 2.6	45
4.		1.5	-	1.0	-	-	-	24^*** ± 1.5		51.6 ± 7.6	20
5.		2.0	-	1.0	-	-	-	2^** ± 1		17.3 ± 2.5	28
6.		2.5	-	1.0	-	-	-	1.3^NS ± 0.6		1.3 ± 0.57	30
7.		0.5	1.0	-	-	-	-	17.3 ^NS ± 2.5		80 ± 5	25
8.		1.0	1.0	-	-	-	-	12.6 ^NS ± 2		29.7 ± 2.5	45
9.		1.5	1.0	-	-	-	-	2^* ± 1		12 ± 2	50
10.		2.0	1.0	-	-	-	-	2.7^NS ± 0.6		13 ± 2	25
11.		2.5	1.0	-	-	-	-	4^NS ± 1.2		35.7 ± 2.0	25
12.		0.1	-	-	0.1	-	-	2^** ± 1		25 ± 1	20
13.		0.2	-	-	0.1	-	-	25.3^** ± 2.5		96 ± 4	22
14.		0.3	-	-	0.1	-	-	2.3^** ± 1.5		1.7 ± 0.57	25
15.		0.4	-	-	0.1	-	-	28.7^** ± 4.2		95 ± 5	23
16.		0.5	-	-	0.1	-	-	11.7^** ± 1.5		85 ± 5	15
17.		0.5	-	-	0.5	-	-	10^NS ± 1		41 ± 3.6	20
18.		1.0	-	-	0.5	-	-	7.3^NS ± 1.2		21 ± 3.6	25
19.	MS (Half strength)	1.1	0.9	-	-	-	-	10 ± 2	Callus	6.7 ± 0.57	65
20.		1.1	-	1.0	-	-	-	6 ± 1	Callus	2.7 ± 1.15	120
21.		-	1.3	-	-	1.6	-		Callus	20.3 ± 5.5	70
22.		-	-	-	-	0.5	0.5		Callus	88.3 ± 9.7	15
23.		-	-	-	-	0.5	1		Callus	96 ± 4	15
24.		-	-	-	-	0.5	2		Callus	90.3 ± 1.5	15
25.		-	-	-	-	0.5	3		Callus	78.4 ± 2.08	15
^***P = < 0.001
^**P = < 0.01
^*P = < 0.05

Moreover, half-strength MS medium enriched with 1.1 mgL^− 1 BAP demonstrated callus formation when given 0.9 mgL^− 1 IAA and 1 mgL^− 1 IBA in 10 to 12 weeks with a meager percent response (6.7 and 2.7%, respectively; Table 1). BAP or Kn coupled with IBA, IAA, or 2,4-D (1.0 + 1.0 mgL^− 1) was studied for callus initiation. Callus development was more effectively produced (100%) by the combination of half MS medium enriched with Kn + 2,4-D (0.5 mgL^− 1 + 1 mgL^− 1) within 6–8 weeks (Table 1). The callus was moved to MS medium supplemented with BAP (1.1 mgL^− 1), IBA (1.0 mgL^− 1), or IAA for shoot regeneration (0.9 mgL^− 1). The maximum shoot regeneration from the callus tested was achieved by BAP (1.1 mgL^− 1) coupled with IAA (0.9 mgL^− 1), producing more than 10 shoots/explants (Table 1).

To determine the optimal growth hormone mixture for multiple shoot induction shoots obtained through direct or indirect regenerations were transferred to various growth hormone combinations. For additional multiplication, the shoots switched to BAP in conjunction with 0.5 Kn, NAA (0.1 or 0.5 mgL^− 1), or 1mgL^− 1 IAA. The combination of 2 mgL^− 1 BAP and 0.5 mgL^− 1 Kn produced the most shoots, followed by 0.2 mgL^− 1 BAP and 0.1 mgL^− 1 NAA (Table 2). Regenerated shoots were transferred to a half-strength MS basal medium containing gibberellic acid (GA3) at 0.5 or 1.5 mgL^− 1 for shoot elongation (Table 2). No discernible increase in shoot length was seen when GA₃was present.

Table 2

Effect of growth hormones on multiple shoot induction of in-vitro-grown *Bergenia ligulata*
S. No.	Hormone combination with half-strength MS medium (mgL^− 1)	No of shoots	Percent response (%)	Shoot multiplication response recorded after
1.	BAP (2) + Kn (0.5)	68 ± 7.6	84.67 ± 5.03	6 weeks
2.	BAP (0.2) + NAA (0.1)	26.33 ± 4.7	94.6 ± 4.6	6 weeks
3.	MS + GA (0.5)	3.67 ± 0.57	88.6 ± 3.2	6 weeks
4.	MS + GA (1.5)	10.6 ± 5.0	88.3 ± 2.1	6 weeks
5.	BAP (1.1) + IAA (0.9)	22.3 ± 2.5	93 ± 3	6 weeks

Table 3: Amplification products generated with responding ISSR primers in mother and in vitro established plantlets of Bergenia ligulata

3.2 Rooting: in-vitro shoots

The micro shoot of B. ligulata obtained from various growth hormone combination treatments was transferred to half strength MS medium along with 1 mgL^− 1 IBA in MS media for root induction. After 6–8 weeks, half-strength MS medium showed better rooting (Fig. 1E, G) as compared to media containing IBA.

3.3 Hardening And Survival Of Tissue Culture Plants

The plantlets were taken out of the flask, washed in distilled water to get rid of the agar, and then transferred to test tubes with regular tap water for a week in a greenhouse. The plants were placed in tiny plastic bags filled with sand, and to keep the moisture level stable at first, those bags were maintained within a tray partially filled with water and covered with a transparent plastic pouch. After a week, the plastic bag's outer layer was removed for 15 minutes, and over the following days, that time was gradually raised to 30, 40, and 60 minutes. The plants were totally removed from their plastic covering and left to survive for 15 days after doing this for a week. Afterward, they were placed in containers with peat soil, perlite, and vermiculite (1:1:1). After 5–7 weeks of the plant's translocation to the glass house, an approximate 80% survival rate was obtained (Fig. 1H, I).

3.4 Genetic Fidelity of in-vitro grown Bergenia ligulata plants

The UBC series of 15 ISSR semi-arbitrary primers were all examined, and 10 of them yielded amplifying products that were unique and clear. With an average of 5.3 bands per primer, ISSR primers (10) effectively generated 58 distinct and scoreable bands in the 300–3000 bp range. UBC primers 810, 841, and 866 produced the most and the fewest amplicons, respectively, with a maximum of (7) and a minimum of (2). (Table 3). Bergenia ligulata plants were proven homogeneous when all 58 amplicons generated from 10 ISSR primers were monomorphic compared to the mother plant (Fig. 2).

Establishing an efficient, economic, and repeatable B. ligulata micropropagation protocol is the subject of the current work. The current study tested several hormone combinations with MS media (full and half strength) for direct and indirect regeneration from leaf and petiole explants. MS media fortified with different growth regulators showed differential efficacy in shoot regeneration (Table 1). Explants grown on MS media supplemented with growth hormones such as BAP, NAA, IAA, and IBA responded as shown in Table 1.

Cytokinins are essential for plant development because they promote the formation and growth of shoot buds by elongating and expanding cells (Mok and Mok 2001). Numerous studies examined the impact of BAP on multiple shoot induction and regeneration in various medicinal plants (Ashraf et al., 2014; Sharma et al., 1993; Singh and Pandey, 2019; Rafi et al., 2016). Our findings in the current study showed that the combination of IBA (1 mgL^− 1) and BAP at a lower dosage of 0.5 mgL^− 1 resulted in direct regeneration from leaf and petiole explants.

Our results are in line with the findings of Singh and Pandey (2019) who evaluated the direct regeneration potential of B. ligulata leaves on MS medium supplemented with 1.1 mgL^− 1 BAP + 0.9 mgL^− 1 IAA. Some researchers also reported that a combination of BAP and IAA was proved to be more effective in Azadirachta indica, Scutellaria Alpina, and Quercus Serrata (Grzegorczyk-Karalak et al. 2016; Pandey et al. 2018).

While analyzing the organogenesis in Hypericum frondosum, the authors found that a combination of 1.1 mgL^− 1 BAP with 0.4 mgL^− 1 IAA, increased the shooting response and multiplication in the plant. Effects of combined growth hormone factors with MS media supplemented with BAP and NAA were examined and observed that 0.4 mgL^− 1 BAP resulted in 28 shoots when combined with the 0.1 mgL^− 1 NAA in two weeks of inoculating the leaves. Our results are as per the findings of Balilashaki et al. 2015, in which authors observed the best combinations of 5 mgL^− 1 BAP with 2 mgL^− 1 NAA for direct shoot regeneration in Phalaenopsis cv. Surabaya.

The current study also showed how several phytohormone combinations affected the growth of shoots, with the suitable combination being 0.2 (mgL^− 1) BAP and 0.5 (mgL^− 1) Kn producing 50 to 60 shoots in 6–8 weeks. In comparison to BAP or Kn alone, various research that looked at the interaction between BAP and Kn concluded that this combination was the best phytohormone combination (Ashraf et al. 2014; Shirin and Rana, 2007). Other phytohormone combinations, notably (0.4 and 0.5 mg L-¹) BAP with 0.1 mg L^− 1) NAA, were nevertheless shown to be highly effective for increasing the number of shoots. According to Rasool et al. (2009), combinations of BAP and NAA promoted the proliferation of shoot regeneration in Prunella vulgaris, which is consistent with the current study's findings.

On a Bergenia leaf/petiole explant, the effects of various 2,4-D doses have also been tested. It was shown that within 15 days of inoculation, a group of callus cells began to form on 0.5 mgL^− 1 Kn with 1 mgL^− 1 2,4-D with half MS with 100% response. However, the percent response of callus induction decreased by 94, 90, 78, and 55, respectively, when the concentration of 2,4 D rose to 2,3,4, and 5 mgL^− 1. The combination of 0.5 mgL^− 1 Kn and 1 mgL^− 1 2,4-D produced the best response (100% response). Bergenia plants can grow new leaves directly from explants, as seen by the SEM photos. Small, numerous shoot buds could be observed growing from the leaf's surface.

An efficient and cost-effective protocol for Bergenia regeneration was devised in the current work, allowing the plant to be propagated within 6 months with a very productive result of more than 3500 plants per explant, whether by direct or indirect regeneration methods. Both the petiole and the leaves served as explants for the investigation. The best combinations for indirect regeneration were found to be 0.5 mgL^− 1 Kn with 1 mgL^− 1 2,4, D on either of the explants (leaf/petiole), whereas the best combinations for direct regeneration were found to be 0.2 and 0.4 mgL-1 BAP with 0.1 mgL^− 1 NAA. The plants were also examined for the ability to induce multiple shoots, and the optimal combination was found to be 2 mgL^− 1 BAP + 0.5 mgL^− 1 Kn. Overall, using leaf and petiole explants, this work provides an effective micropropagation method for Bergenia ligulata species.

BAP: Benzyl aminopurine, NAA: Naphthaleneacetic Acid, Kn: Kinetin, IAA: Indole Acetic Acid, 2,4-D: 2,4-Dichlorophenoxyacetic acid

Acknowledgments

The author is grateful to the Director CSIR-CIMAP for providing basic infrastructure, instrumental facilities, and lab space for conducting the experiments. The author is also thankful to the Department of Science and Technology, New Delhi for providing Women Scientist Fellowship-A (SR/WOS-A/LS-226/2018) for the project based on Bergenia ligulata.

Statements and declaration

The authors declare no competing financial interests or personal relationships that could affect the results reported in this article.

Authors Contribution

FD carried out the experiment, recorded the data, and prepared the manuscript, LR designed the experiment and reviewed the manuscript, PS did the SEM studies, MK performed the genetic fidelity testing, and ZIW assisted in executing the experiment.

Data Availability

The published article contains all the data generated and analysed during the study (No supplementary data included)

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Micropropagation studies in Bergenia ligulata: A potential plant remedy to kidney stones

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Abstract

Figures

Key Message

Introduction

Experimental Plant Material

2.2 Surface Sterilization And Culture Conditions

2.4 Microscopy (Sem Studies)

Molecular Analysis

2.5 Extraction and quantification of Genomic DNA

2.6 Amplification Through Pcr

2.7 Pcr Product Validation

2.8 Statistical Analysis

Results

3.3 Hardening And Survival Of Tissue Culture Plants

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

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