IT’S ALL ABOUT TECHNIQUE: IN VITRO METHODS FOR STORING MEDICINAL PLANT GERMPLASM

By Rustin A. Rohani, MA, BSc

Despite the arrival of pharmaceutical drugs, humans have historically used plants to treat illnesses (Ganie et al., 2015). Even today, there are about 30 essential drugs directly sourced from plants (Veeresham, 2012). There are almost 18,000 documented medicinal plants in the world with many of them threatened from climate change and land usage (Willis & Bachman, 2016). The World Health Organization (WHO) reports that the majority of the world’s human population still relies on medicinal plants and that health-policy makers continue to recognize how important medicinal plants are (WHO, 2002). For example, secondary metabolites in some medicinal plant extracts can act as antioxidants to treat female reproductive disorders such as infertility, amenorrhea, dysmenorrhea, gonorrhea, hemorrhages, menstruation unrest, and inflammation of the ovaries (Mbemya et al., 2017). Also, researchers at Golestan University of Medical Sciences, Pasteur Institute, Payame Noor University and University of Karachi have identified anticoagulant compounds in some herbal species of plants that can be used to prevent blood clotting and stroke (Aalikhani Pour et al., 2016). With studies such as these showing the importance of medicinal plants for medicinal uses, it’s vital to eff ectively preserve them, because the eff ectiveness of the natural elements found in many herbal plants are dependent on how these plants are collected and stored (WHO, 2002).

Collecting and storing seeds has been a common and effective ex situ method of preserving plant genetic resources, but there are reasons why this may not be the best method. Some plants do not produce enough seeds due to low fecundity, so banking in vitro tissues becomes vital (Pence, 2011). In addition, seed banking can increase susceptibility to diseases, and seeds may lose the capacity to germinate after a period of time (Jha, 2015). In contrast, the advantages of using in vitro methods to store plant germplasm (plant genetic material) involve tissue culture collections being disease-free and controlled in a protected ex situ environment (Mycock, Blakeway, & Watt, 2004).

According to Thorpe (2007), plant germplasm storage has been discussed and applied through developed in vitro techniques since the mid-20th century. Developed techniques have mainly involved plant growth retardants on plant growth media, low temperatures, and cryopreservation through liquid nitrogen storage (Thorpe, 2007). These in vitro techniques have shown to be a viable option in storing plant genetic resources, including medicinal plants.

PLANT GROWTH RETARDANTS ON PLANT GROWTH MEDIA

When it comes to storing plant germplasm, cultivating plant tissue through in vitro processes is required. Two main components of cultivating plant tissue are using plant growth medias and plant growth retardants (PGRs). Using PGRs, such as auxins and gibberellins, allows for natural plant hormones to be blocked when cultivating plant tissue on plant growth medias (Thorpe, 2007). Gibberellic acid is the specifi c hormone PGRs are used to block, because this hormone provokes plant cell growth (Thorpe, 2007).

The benefits of using PGRs to inhibit plant growth include more storage space (regulated plant stretch) and aiding in the suppression of abnormal growth (plant disease) such as overgrowths, leaf epinasty, and superfluous branching (Getter, 2013). A major plant growth media used with PGRs is Murashige and Skoog media (MS), which is made up of major salts, minor salts, vitamins, and natural ingredients (Trigiano & Gray, 2010). The primary disadvantage to using plant growth media, such as MS, is that routine sterilization is necessary to prevent any pH diff erences, contamination and/or degradation of the media (Saad & Elshahed, 2012).

However, a sixteen week slow-growth mid-term conservation study done by Chauhan et al. (2016) showed a 100% survival rate for the microshoots of an endangered herbal plant species (Chlorophytum borivilianum) when MS via PGRs was used. Chlorophytum borivilianum is an important herb because of its treatment uses on diabetes and joint-related diseases (Chauhan et al., 2016). Similarly, researchers from Al-Balqa Applied University stored microshoots from another herbal plant, Stevia rebaudiana, for thirty-two weeks with a 93% survival rate using the same media with diff erent PGRs (Shatnawi et al., 2011).

Although it is clear that there are risks to the effectiveness of plant growth media if periodic sterilization is not appropriately executed, still, high survival rates of these cultivated medicinal plants by MS via PGRs proves that the rewards outweigh the risks. To minimize risks, researchers may practice eff ective laboratory management strategies, such as sterilization protocols through autoclaving laboratory equipment.

According to Sigma-Aldrich (n.d.), plant growth medias should normally be autoclaved at 121 degrees Celsius for proper sterilization. It is also important to take into account that greater volumes of media require a longer time to sterilize in an autoclave (Sigma-Aldrich, n.d.).

NON-FREEZING TEMPERATURE STORAGE

Part of storing plant tissue culture involves placing medias under low temperatures. Usually, temperatures between 1 to 9 degrees Celsius are considered nonfreezing in regards to storage of plant material (Thorpe, 2007). The advantages to conserving plant germplasm in cold storage is the simplicity, fi nancial practicality, and long-term survival ability of plant germplasm (Jha, 2015). Cold storage of medicinal plant germplasm is quite advantageous due to its practical and safe approach. Nevertheless, the genetic characteristics of medicinal plant species stored under this method should be further evaluated to ensure consistent survival rates across tested samples.

A study done by Gianni and Sottile (2015), measured the eff ects of cold storage and slow-growth of two species of plum, Prunus domestica L. and Prunus cerasifera, which have medicinal properties. The results of this study proved cold storage to be eff ective for these Sicilian plum species in regards to survival. Under cold conditions, genotypes from these species exhibited nominal growth and had survived between 6 to 12 months. However, the survival rates of both these species in cold storage was inferred to be dependent on their genetic predisposition (Gianni & Sottile, 2015).

Another advantage to non-freezing temperature storage is that plant tissue and cells are not at risk of being injured from cryopreservation, such as plant dehydration due to freezing (Jha, 2015). Scientists have compared both cold storage and cryopreservation methods with medicinal plants by comparing these methods on three species of medicinal plants, which include Eruca sativa Mill., Astragalus membranaceus and Gentiana macrophylla Pall. During a five month cold storage period, 80% to 100% of tissue samples from all three species survived (Sheng-Hui et al., 2008). Cryopreservation in regards to these species presented a lower survival rate during this five month period, and plant tissue samples from G. macrophylla expired from freezing temperatures (Sheng- Hui et al., 2008).

CRYOPRESERVATION

Cryopreservation is similar to cold storage technique of plants in that it involves the development of sterile tissue cultures (Jha, 2015). In addition, the plant’s genetic material is treated with cryoprotectants and then stored under temperatures of -196 degrees Celsius (Jha, 2015).

Shatnawi et al. (2011) states that cryopreservation is an advantageous technique for storing medicinal plant germplasm, because very low temperatures cause a restraint of biochemical processes. In consideration of this fact, scientists should still take calculated risks when cryopreserving herbal plant genetic resources in order to store a suffi cient amount of collected samples.

The application of cryoprotectants on microshoots, plantlets, embryos, and cell culture, allows for storage through (liquid nitrogen) cryo freezing. According to Tao and Li (1986), cryoprotectants are compounds categorized by molecular weight that inhibit freezing injury to plant tissue. The higher the molecular weight the cryoprotectant has, the more likely these compounds will cause damage to the plant’s cells. However, concentrations of high molecular weight polymers are necessary, like dimethyl sulfoxide (DMSO), in order to penetrate the plant’s cells and be effective. They suggest that a combination of cryoprotectants with differing molecular weights may lessen the degree of damage to the plant’s cells (Tao & Li, 1986). Yet, experimenting with cryoprotectants may be an insufficient method when endangered medicinal plants are involved.

Inventor Meryman (1997) argued that the use of penetrating cryoprotectants and controlled freezing are not efficient techniques for cell recovery. This is due to the risks involved with the use of cryoprotectants on cells, such as the risks of ice growth during rewarming of plant tissue and the impracticality of consistent cooling and rewarming procedures of large sample sizes (Meryman, 1997). It’s also important to consider the costs of time and financial resources of experimentation.

IN-VITRO, COST, AND FUTURE

In review, in vitro techniques are viable alternatives to storing medicinal plant genetic resources. As far as PGRs on plant growth medias, sterilization is an important aspect that can affect the technique in cultivating plant germplasm. The slow growth storage of plant germplasm is shown to be practical financially and procedurally. Lastly, freezing plant germplasm under cryopreservation involves risks, such as plant tissue or cell damage. Yet, survival rates under this in vitro method have been proven to be high.

From these techniques, institutions should also factor economic cost. Expenses to consider for in vitro storage of medicinal plants include aseptic equipment, laminar fl ow cabinets, and labor (Pence, 2011). Specifically, for cryopreservation of plant genetic resources, materials such as a liquid nitrogen storage tank is also necessary (Pence, 2011). Furthermore, scientists at the National Laboratory for Genetic Resources Preservation state that the viability of cryopreserved collections may be statistically dependent on the number of plant genetic resources stored and the confidence levels of the researchers (Volk et al., 2017).

Biobanking plant genetic resources can be further studied and supported through in vitro methods not directly involved in storage. For example, the polymerase chain reaction (PCR), allows for in vitro amplification of medicinal plant DNA. In fact, reviews of this technique have shown that molecular markers can provide information regarding the genetic diversity and genetic makeup of herbal plants at a molecular level (Sarwat et al., 2012).

In addition, technology has allowed scientists to characterize and assist in conserving plant genetic material through molecular computing (Thorpe, 2007). In particular, in silico methods, such as cheminformatics, have allowed researchers to identify chemical compounds in medicinal plants by using computational techniques. Cheminformatics has helped researchers deduce herbal plants by chemical compounds that are most effective for treatment of diseases (Aalikhani Pour et al., 2016).

CONCLUSION

It is important to consider that there are endangered medicinal plant species in many parts of the world, and storing their germplasms should be prioritized as a result. The International Union for Conservation of Nature, an international organization associated with conserving natural resources, lists some medicinal plants that are threatened due to overexploitation and are known to treat diabetes, digestive disorders, and certain cancers (IUCN, 2016). These plants include Cistanche deserticola, Dioscorea deltoidea, Nardostachys grandiflora, Picrorhiza kurrooa, Pterocarpus santalinus, Rauvolfi a serpentina, and Taxus wallichiana (IUCN, 2016). By using a combination of both in vitro and in silico methods, researchers can invest more time into storing medicinal plant germplasm that can be most benefi cial to treating various illnesses for humankind.

Rustin Rohani completed this project as a part of his graduate work with Project Dragonfl y at Miami University in Oxford, Ohio.

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