Research Article

Liquid Culture System for In Vitro Propagation of Physalis minima L. a Threatened Medicinal Plant

Muhammad Akram¹,²*, Rashid Mahmood³, Fahim Arshad4 and Umer Farooq Gohar5

¹Department of Biology, Government Shalimar Graduate College, Lahore, Punjab Higher Education Department, 54000 Lahore, Pakistan; ²Institute of Botany, University of the Punjab, Lahore-54590, Pakistan; ³Department of Botany, Government MAO College, Lahore, Punjab Higher Education Department, 54000 Lahore, Pakistan; 4Department of Botany, University of Okara, Okara-56300, Pakistan; 5Institute of Industrial Biotechnology, Government College University, Lahore-54000, Pakistan.

Received | December 20, 2022; Accepted | April 03, 2023; Published | April 10, 2023

*Correspondence | Muhammad Akram, Department of Biology, Government Shalimar Graduate College, Lahore, Punjab Higher Education Department, 54000 Lahore, Pakistan; Email: [email protected]

Citation | Akram, M., R. Mahmood, F. Arshad and U.F. Gohar. 2023. Liquid culture system for in vitro propagation of Physalis minima L. a threatened medicinal plant. Journal of Innovative Sciences, 9(1): 72-82.

DOI | https://dx.doi.org/10.17582/journal.jis/2023/9.1.72.82

Keywords | Callus, Wild gooseberry, Liquid culture system, Medicinal plants, Physalis minima, Micropropagation, Thidiazuron

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction

Medicinal plants are a natural source of bioactive ingredients, which are used in both curative and preventive medical therapies (Timalsina et al., 2021; Kim et al., 2017). More than 80% of the world population has been estimated to rely on medicinal plants or their derivatives for common healthcare needs, in complementary and traditional medicines (Mbuni et al., 2020). Specifically, this is more common in third world countries (Mbuni et al., 2020; Timalsina et al., 2021; Umar et al., 2021), where access to traditional drugs based on modern healthcare is scarce and costly (Popovic et al., 2016). The sick people typically use combinatorial approaches to gain elevated therapeutic effects from complementary and traditional drugs (Gagnier et al., 2007). Moreover, the medicinal plants are consumed as an important component of daily intake, which confer functional benefits to activating biological processes (Bansal and Chhibber, 2010; Sharifi-Rad et al., 2020). This has accelerated the demand for plant-derived herbal drugs, natural health products, and secondary metabolites even in the developed world. Exploitation of medicinal plants in developing traditional drugs is therefore on the rise (Sheeba et al., 2010; Intzaar et al., 2013).

Physalis minima is commonly known as wild gooseberry, is an important medicinal plant of the family Solanaceae (Sheeba et al., 2010). It is a perennial herb 20–50 cm tall in size (Durga et al., 2020), and is frequently found in organic matter-rich waste as well as agrarian land, distributed around the tropical and subtropical regions of Asia (Chothani and Vaghasiya, 2012). It holds strong medicinal potential, as a diuretic and laxative, and has been used as a treatment for multiple conditions including splenomegaly and ulcer of the bladder (Canh et al., 2021). Crushed leaves and fruits are used to neutralize snake bites (Karthikeyani and Janardhanan, 2003), and its plant extract possess anti-inflammatory and anticancer activities (Khan et al., 2009). Sadly, P. minima has begun to regress sharply, particularly in economically depressed regions (Intzaar et al., 2013). This is mainly due to its suboptimal seed germination (Singh, 2009), the extensive use of herbicides (Vaverkova et al., 1995a, b), and the lack of modern cultivation practices among rural farmers in developing countries (Ahmad and Habib, 2014). Enhancing survival of P. minima through creating new conservation strategies is imperative. Short-term conservation strategies such as awareness campaigns among farmers are planned, however, in vitro multiplication approaches are needed in the long-term, serving as a promising alternative strategy for rapid conservation of P. minima.

In vitro plant tissue culture technology is a suitable alternative to enhance the survival of medicinal plants (Afroz et al., 2009; Intzaar et al., 2013). It is based on seeding any tiny piece of the target plant on a defined growth medium, producing a large number of calli, which then have ample potential to be transformed into numerous highly proliferative shoots and adult plants (Jones et al., 2007). A supportive growth medium is requisite for effective callus induction and requires a watchful supplementation of plant growth regulators. Thidiazuron (TDZ ) is a strong growth regulator, which has cytokinin and auxin-like properties for different morphogenic responses (Murthy et al., 1998). It plays a substantial role in in vitro seed germination and somatic embryogenesis in multiple herbaceous and woody plants (Akram and Aftab, 2015a, b; Akram and Aftab, 2016). The concentration of TDZ is plant specific (Dewir et al., 2018; Xiong et al., 2022; Dalavi et al., 2023), and it is therefore imperative to explore an appropriate concentration of TDZ that could support in vitro propagation of P. minima while preserving its normal growth characteristics.

Thus, objective of the present investigation was to optimize in vitro propagation method for mass multiplication and conservation of P. minima using TDZ in a liquid medium.

2. Materials and Methods

2.1 Procurement of plant material and culture conditions

Leaves of wild P. minima were collected from the turmeric field crop located in village Dhing Shah Tehsil and District Kasur, Punjab, Pakistan in September. Juvenile leaves were collected from top of the plant brought to Plant Tissue Culture Lab and thoroughly washed under running tap water. Plant was dipped in 1% detergent solution (w/v) (Ahmad et al., 2022) (Bright, Colgate Palmolive, Pakistan) for 10 minutes and then sanitized using 10% (v/v) Robin Bleach (Rekitts and Benkiser Group, Pakistan). Such explants were then washed with sterile water under cabinet chamber. The culture medium was autoclaved (Labtron, LVA-F22 UK) at 121 °C and 104 kilo Pascal pressure for 15 minutes and incubated at room temperature (25±2°C) under defined culture conditions for 16 hours photoperiod (35 µmole m-1s-1 provided with white fluorescent tube light, Philips). Leaves were inoculated on the sterilized culture medium.

2.2 Callus induction

For callus induction, discs of 5 × 15 mm2 leaf explants were cultured (abaxial side) on Murashige and Skoog (1962) (MS) basal medium. One leaf disc explant was used per culture vessel per treatment. Ten replicates/culture vessels were used for each treatment and the experiment was repeated thrice. Solution was prepared by dissolving the salts separately, which was then fortified with 0.1, 0.5, 1.0, 2.0 or 5.0 µM Thidiazuron (TDZ ) (Sigma Aldrich, USA) for callus induction. At day 14, the rate of callus induction and morphology were collected.

2.3 Shoot regeneration

A one-gram callus was used per culture vessel containing MS liquid as well as agar-based (Agar, Phytotechnology Labs) solid medium devoid of any plant growth regulators (PGRs) in three tissue culture vessels including 150 mL capacity glass jars (25 mL active culture volume) (Mitchells), 25 × 150 mm culture tubes (Pyrex; 10 mL active culture volume), and 250 mL capacity Erlenmeyer flasks (50 mL active culture volume). All cultures were sub-cultured after every 15 days on the liquid and solid medium. Shoot culture from the solid medium was shifted to fresh medium after 15 days. Shoot regeneration percentage and total number of shoots per callus were recorded after 15 days of initial culture. A complete disappearance of chlorophyll in the shoot culture was regarded as an albino phenotype.

2.4 Regeneration of albino shoot cultures

Clumps of albino shoots, including some basal callus portions, were inoculated in a 2-liter capacity Erlenmeyer flask containing liquid MS basal medium (150 mL active culture volume). The Erlenmeyer flask was capped with aluminum foil. The 2 Syringe Millipore filters (SMF) were adjusted in the caps of flask with one SMF for medium inlet and the 2nd for aeration. After every 15 days, fresh MS liquid and solid media were added to the Erlenmeyer flask. Results of shoot number per flask (10 total number of flasks) was recorded after thirty, sixty, ninety and one-twenty days of initial culture. Shoots numbers were recorded without removing the shoot cultures from the Erlenmeyer flask.

2.5 Root induction and hardening

Rooting of shoots was carried out in half strength MS medium supplemented with indole-3-butyric acid (IBA) or Naphthalene Acetic Acid (NAA) (Sigma) @ 1, 2, 3 or 4 µM. Rooting percentage, number and length of roots were observed after 10 days of culture. Roots were immersed in 1% solution of Dithane fungicide (M45, Corteva) and shifted in plastic pots with 1:2 ratio of garden soil and peat moss. After 35 days, the rate of plantlet survival was recorded and transferred to the field conditions.

2.6 Data analysis

A completely randomized design was applied for data collection. The data was evaluated using ANOVA and Duncan’s multiple range test (DMRT) to determine statistical difference between the means of any two treatment groups at p<0.05. Binomial data were evaluated via logistic regression. Poisson regression was used for count data. R2 where applicable was used to determine Polynomial contrast. At least ten replicate cultures per treatment group were used and every experiment was repeated thrice using SPSS v 16.0. (SAS, USA).

3. Results and Discussion

3.1 Callus induction

Thidiazuron (TDZ) has been shown to promote efficient callus induction, therefore we first investigated effective concentrations of TDZ in Murashige and Skoog (MS) basal medium to support P. minima callus induction. For this, the medium was supplemented with five concentrations of TDZ (0.1, 0.5, 1.0, 2.0, 5.0 µM) and callus induction was carried out (Figure 1) from juvenile leaf explants (Figure 2A). Swelling of the explants was observed after 6 days of culture, which was followed by the initiation of callus from the cut margins of the explants across the five culture conditions (Figure 1B). The callus induction rate was measured at day 14 of the initial culture and data was evaluated using the polynomial contrast approach, which revealed a significant correlation between callus formation and TDZ 1.0 µM (Pearson coefficient of determination R2 = 0.8697, p = 0.001). 100% callus formation was observed on medium containing 1.0 µM TDZ (Figure 1A). However, callus formation began to decline at 2.0 µM or higher TDZ (Figure 1A). It is noteworthy that the morphology of calli was different in different TDZ concentrations (Figure 1B-H). While 0.1-0.5 µM TDZ produced whitish and granular calli (Figure 1B-E), the 1.0 µM or higher concentration of TDZ resulted in nodular and morphogenic green calli (Figure 1F-H). This data showed that TDZ supplementation could result in varied morphogenic effects on P. minima calli, and the 1.0 µM appeared optimal for maximal callus formation in the span of 14 days.

 

3.2 Shoot regeneration and proliferation

One gram of the 14-day old callus culture was transferred from the 1.0 µM TDZ concentration to the liquid or agar-based solid MS basal medium (Table 1) in three culturing vessels including Erlenmeyer flask, glass jar, and culture tube (Table 2 and 3) for shoot regeneration. Shoot regeneration initiated within 5 days of callus transfer in both liquid and solid medium in all culturing vessels (Figure 2B and 2C; representative photos). Notably, shoot regeneration rate and numbers were significantly higher in the liquid medium as compared to the solid medium at 15 days post culture transfer (99.25% with 35.00 shoot numbers versus 82.12 with 25.45; also see Table 1). The shoot regeneration appeared more pronounced in glass jars (99.25%) than the culture tubes (95.33%) and the Erlenmeyer flasks (80.23%) (Tables 1 and 3). The agar-based solid medium also supported shoot regeneration albeit with significantly lower regeneration rate and shoot numbers (Table 1). Interestingly, subculturing of the shoot culture before or at day 15 could maintain the proliferation propensity for up to 12 months with no visible morphological abnormality observed in the shoot regeneration or proliferation as demonstrated in Figure 2 C. Together, this data suggested that 1.0 µM TDZ supplementation to the MS basal medium was sufficient to prime leaf explant derived calli to display elevated potential in shoot regeneration and proliferation in basal liquid medium.

 

Table 1: Shoot regeneration from callus cultures induced by 1.0 µM TDZ after 15 days of P. minima.

MS Medium	Shoot regeneration (%)	Number of shoots
Liquid	99.25±6.32a	35.00±4.25a
Agar	82.12±4.25ab	25.45±2.45b
Regression	p = 0.03221	p = 0.00112

 

Data is presented as mean values (±SE). Letters in the superscript in rows represent significant difference amongst the means of the two groups evaluated by the ANOVA followed by DMRT. At least 10 replicate culture of three repetitions. ¹Logistic regression, ²Poisson regression.

 

 

3.3 High frequency shoot formation

The propensity of shoot regeneration and proliferation

 

Table 2: P. minima shoot multiplication from the albino shoots in the basal liquid culture.

MS basal medium	Number of multiple shoot formation in Erlenmeyer Flasks
	30 days	60 days	90 days	120 days
Liquid medium	54.60±11.32d	138.75±14.36c	345.32±62.35b	652.78±25.66a
Solid medium	34.12±14.25d	145.32±32.25c	241.02±27.32ab	352.78±45.23a

 

Data is presented as mean values (±SE). Letter in the superscript in rows represent significant difference amongst the means of the two groups evaluated by the ANOVA followed by DMRT. At least 10 replicates with 3 repetitions were carried out.

 

Table 3: Shoot regeneration from callus cultures induced by 1.0 µM TDZ after 15 days of P. minima.

MS medium	Erlenmeyer flasks		Culture tubes
	Shoot regeneration (%)	Number of shoots	Shoot regeneration (%)	Number of shoots
Liquid	80.23±5.23a	19.12±3.25a	95.33±5.78a	18.25±4.25a
Agar	75.75±4.12b	16.23±4.25ab	76.52±6.32b	14.33±5.65ab
Regression	p=0.02211	p=0.00012	p=0.03301	p=0.00212

 

Mean values (±SE) followed by different letters are significantly different according to Duncan’s multiple range test p<0.05. Each value is the mean of 10 replicates and the experiment was repeated thrice. ¹Logistic regression, ²Poisson regression.

 

 

remained stable for 15 days without refreshing the medium, but beyond this, the albinism prevailed with shoots displaying visible chlorophyll loss (Figure 3A). We then tested whether the albino shoots derived from the 1.0 µM TDZ-primed calli could be rejuvenated to high frequency shoot formation. For this, 20-days old albino shoots were invigorated

in fresh MS liquid medium in an Erlenmeyer flask, with solid medium used as a control. Intriguingly, multiple shoot buds appeared as early as 10 days of invigoration, which transformed into lush green healthy shoots (Figure 3B). The liquid medium produced significantly more green shoots (54.60) in the albino shoot culture than the solid medium (34.12) at 30 days of rejuvenation. Moreover, the number of shoots continued to increase over time and reached 138.75, 345.32 and 652.78 at 60, 90, and 120-days of rejuvenation, respectively (Table 2). It is noteworthy that a similar increasing trend of shoot formation and multiplication over time was observed in the albino shoots cultured on the solid medium (Table 2). This further pointed to the existence of elevated shoot regenerative potential imparted by the 1.0 µM TDZ priming during callus induction. We also tested albino shoot regeneration in the glass jar and culture tube culturing vessels (Table 2). However, shoot formation frequency was high in only the Erlenmeyer flask, possibly due to adequate nutrient diffusion and aeration in the spacious vessel (Table 2). Overall, shoot formation and multiplication remained visibly normal throughout experiments (Figure 3C), however, some minor signs of hyperhydricity were noted in a few cultures grown in the liquid medium (35.33%) (Table 4).

 

Table 4: Shoot hyperhydricity prevalence in different culture media the Erlenmeyer flask. The culture was revised from the albino shoot of P. minima.

MS medium	*Hyperhydricity (%)
Liquid	35.33 ± 4.36
Agar	25.36 ± 3.25

 

 

3.4 Root induction and hardening

The proliferating shoots (Figure 2 and 3) derived from the 1.0 µM TDZ-primed callus (Figure 1) were rooted in half strength MS medium + 1.0, 2.0, 3.0 or 4.0 µM either IBA or NAA. The rate of rooting was highest (95.45%) with 10.78 number of roots and 55.85 mm root length was observed in medium containing 1.0 µM IBA (Figure 4A) as compared to the medium containing the same concentration of NAA, which provided 70.21% root induction with 10 mean number of shoots and 55.74 mm root length after 35 days (Figure 4B). Plantlets acquiring 95% roots (Figure 5A) were shifted to the glasshouse for acclimatization. They were then further planted in field conditions. The fully acclimatized adult plants started to produce flowers and fruits successfully at the age of 35 days (Figure 5B).

Medicinal plants play a crucial role in socio-cultural, healthcare, and pharmaceutical industries all over the world (Mbuni et al., 2020). Poor seed germination (Singh, 2009), massive use of herbicides (Vaverkova et al., 1995a, b), and over-harvesting of fields by farmers have caused significant threat to the existence of medicinal herbaceous plants, including P. minima (wild gooseberry). Towards addressing this problem, the present study has developed a straightforward in vitro tissue culture approach as a long-term conservation strategy to preserve wild gooseberry plant from going extinct (Canh et al., 2021; Wu et al., 2020). This strategy is based on efficient callus induction mediated by a low concentration of thidiazuron (TDZ; 1.0 µM) in basal MS medium. This initial TDZ-priming appears essential as it promotes effective shoot multiplication of P. minima in the plant growth regulator-free basal medium (Anjani and Kumar, 2018). However, the underlying mechanism as to how TDZ-mediated callus induction exhibits high potential for shoot regeneration and proliferation remains elusive. Thidiazuron is reported to be stable (Mok et al., 1982), and its metabolism is extremely slow in plant tissue culture (Mok et al., 1985). Additionally, it is likely that the remnants of TDZ-derived metabolites play a supporting role (Mok et al., 1985). Indeed, two new TDZ derivatives (1-[1, 2, 3] thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl) urea (3FMTDZ) and 1-[2-(2-hydroxyethyl) phenyl]-3-(1, 2, 3-thiadiazol-5-yl) urea) have been identified as being very potent inhibitors of cytokinin oxidase/dehydrogenase (Nisler et al., 2016) accumulate in plant tissues via suppressing the activity of cytokinin oxidase (Horgan et al., 1988; Hare et al., 1994), and accumulated cytokinins can in return promote shoot multiplication. Also, TDZ modulation of gibberellin biosynthesis has been proposed too (Ali et al., 2022). However, unbiased metabolomic investigations as done before (Erland et al., 2020) focusing on different stages of micropropagations of P. minima are warranted for further clarification in future studies.

Previous efforts in this field have focused on propagating P. minima through artificial means, which includes in vitro synthetic seed production and growth induction by low temperature and osmotic agents of sucrose, mannitol, and sorbitol in tissue culture medium (Yücesan et al., 2015; Nasiruddin and Islam, 2018; Rezende et al., 2018; Yadav et al., 2019). While application of such methodologies could serve the short-term conservation needs of P. minima, the present method ensures long-term culture survival for up to 12 months by refreshing medium within 15 days, thus providing the foundation for a long-term conservation strategy for P. minima (Figure 3C).

Rapid shoot regeneration has a direct relationship with shoot organogenesis, which has been reported previously to respond variably to liquid and solid media (Jones et al., 2007; Afroz et al., 2009; Intzaar et al., 2013). We and others have investigated multiple aspects of shoot organogenesis in liquid and solid media, with the shoots of woody (Akram and Aftab, 2016) and medicinal (Hailu et al., 2022) plants tending to grow longer and quicker in liquid medium. The abundant shoot formation of P. minima in the present study is therefore consistent with previous reports describing the efficacious role of liquid medium (Akram and Aftab, 2016; Hailu et al., 2022). Moreover, shoot regeneration is more pronounced in the Erlenmeyer flask, which is also in line with previous reports describing how culturing vessels with larger volume are effective for fast shoot formation in liquid medium (Kaçar et al., 2010). This could also be due to more extensive physical contact between medium and shoots, which may enhance nutrient uptake and gaseous exchange between medium and shoot biomass (Choi et al., 2001).

We noticed that the shoots derived from the TDZ-primed calli begin to turn albino when medium was not refreshed at day 15 of shoot regeneration. Albinism often reflects differences in genotype, environmental conditions, or genome-based modifications that are reported to be associated with TDZ treatment (Kumari et al., 2009; Lin and Chang, 1998). Interestingly, the shoot albinism appearing in the present study does not seem to be associated with TDZ treatment, as re-greening of albino shoots was promptly achieved by invigorating them in fresh basal medium. This suggests that the prevalence of the albinism may not be a consequence of TDZ treatment. Rather, it could well be associated with nutrient depletion in the culture medium and can swiftly be reversed upon supplying nutrient-rich medium. The reason behind this complete reversal of shoot albinism in P. minima remains puzzling and will require more investigation in future endeavors. However, it is possible that culture refreshed with sucrose could switch on some chlorophyll synthesis pathways resulting in rejuvenation of albino shoots and abundant shoot formation (Wolff and Price, 1960). Alternatively, TDZ-priming may induce a shift in metabolic reprograming in the calli leading to reduced requirement for nutrients, and a survival of shoot culture even in the absence of chlorophyl. This is consistent with the notion that TDZ treatment could result in the accelerated transcription, in vitro, of systems containing chromatin and RNA polymerase I from TDZ-treated leaves (Karavaiko et al., 2004). Realization of such a shift in metabolic reprogramming in in vitro propagation of P. minima is highly speculative and deserves further investigation at the genomic level.

The present study encountered two challenges; firstly, marginal contamination events were observed during initial commencement of experiments including callus induction. This issue was curtailed via improving and strictly adhering to the recommended aseptic guidelines for plant tissues (Singh, 2018; Durul and Memis, 2022). Overall, 90% of our culture remained free from any contamination (Table 3). Secondly, like others (Kevers et al., 2004; Martinez and Deklerk, 2010), this study also encountered the hyperhydricity challenge, which was confined to only shoots growing in liquid medium. This phenomenon was pronounced when the cultivation time exceeded 120 days (35%; Table 4). Future endeavors will be made to reverse the hyperhydricity issue by optimizing liquid medium components, possibly through controlled supplementation of cobalt and silver salts. Recently, addition of a combination of CoCl2 and AgNO3 salts has been shown to completely inhibit hyperhydricity without compromising shoot numbers (Sreelekshmi and Siril, 2021). Additionally, beneficial effects of custom-made culturing vessels with elevated aeration via agitation could be explored, as proper aeration of the culture medium is negatively corelated with hyperhydricity appearance (Reyes-Vera et al., 2008).

In conclusion, the present study demonstrated 100% callus induction from juvenile leaves explants of P. minima in a MS basal medium containing 1.0 µM TDZ. Importantly, TDZ-primed calli exhibited ample potential for efficient shoot regeneration and proliferation in the MS liquid medium devoid of any plant growth regulators. Moreover, emerging shoots from TDZ-primed calli are lush green, flourishing promptly in spacious Erlenmeyer culturing flasks. These shoots were effectively rooted in ½ MS basal medium supplemented with 1.0 µM IBA. The plantlets were then able to be acclimatized and transferred to the field successfully. In summary, the present protocol provides a simple and economical in vitro liquid culture system for massive propagation of P. minima, thus setting the stage for developing a long-term conservation strategy for P. minima that could be employed for the other multiple endangered species of medicinal plants.

Acknowledgments

The first author is thankful to University of the Punjab, Lahore, Pakistan for the provision of research facility for this study.

Novelty Statement

Albino shoots reverted back to green shoot clusters after invigorating with fresh medium.

Author’s Contribution

Muhammad Akram; experimental work and write-up.

Rashid Mahmood; editing and proof reading.

Fahim Arshad; statistical analysis.

Umer Farooq Hohar; Technical assistance.

Conflicts of interest

The authors have declared no conflict of interest.

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