Ishan Herath
Age 17 | St. Catharines Ontario
Winner of the “Best in the Fair” Trophy, Gold Medal Winner Senior Category, Brock University Science Fair Scholarship, Ontario Horticulture Association - District 9 Award, Niagara Peninsula Fruit & Vegetable Growers Association Award, Vineland Research and Innovation Centre Award, University of Guelph Plant Sciences Award, Niagara Peninsula Conservation Authority Award
Introduction:
Vincetoxicum rossicum, commonly known as Dog-strangling vine (DSV) is a perennial herb introduced to North America from the Ukraine and Russia. Over the last 105 years, it has spread exponentially across southern Ontario, southern Quebec and in several north-eastern states of America [Figure 1]. DSV has become an extremely dangerous invasive species (Anderson, 2012; Kricsfalusy, 2008).
Figure 1: Non-linear model for predicting DSV growth shows exponential growth rather than linear growth indicating high risk to the environment. The model coefficients are statistically significant at 99% and R2 is 98%.
DSVs great success can be attributed to several factors, including the ability of the plant to grow in a vast variety of environments, a lack of natural predation due to the toxic alkaloids within its leaves and roots, and its highly effective seed dispersal method. DSV spreads at alarming rates of around 28,000 seeds per square meter (Kempthorne, n.d.). Each plant itself can produce anywhere from 60-80 seed pods, each containing around 40-50 seeds. Each seed is attached to a parachute-like structure called a pappus, enabling them to be carried by the wind, animals, and even by hikers walking along trails. All three methods allow for widely spread seed dispersion and for the formation of dense mats of DSV covering the ground (OMAFRA, 2018).
As a result of DSV’s success, native plant and animal species are being negatively affected. The dense mats of groundcover created by the twining of the DSV vines strangle out the native plants and prevent them from growing and spreading seeds to repopulate. Due to the toxicity of its leaves and roots, and its density of floor covering native herbivorous species cannot eat nor easily traverse DSV. This means that herbivorous species must graze on the small amount of native plants which have escaped DSV, leading to food shortages (Anderson, 2012). Furthermore, monarch butterfly populations have also been severely impacted by the spread of DSV. The monarch butterflies tend to lay their eggs on the toxic leaves of the plant. As a result, the larvae are not able to develop and complete the rest of the hatching cycle and they die from exposure to the toxins.
Figure 2 (a), (b),(c),& (d): Dog-Strangling Vine growing in thick forest floor mats overtaking many native species in Morningside Park, Scarborough, Ontario.
The widespread growth of DSV has led to the prevention of forest regeneration which in turn can result in severe ecological damage that will be very costly to control (Anderson, 2012). These issues are illustrated in Figure 2 which shows Morningside Park, Scarborough, Ontario and its infestation of DSV.
The economic loss due to invasive plants such as DSV is an estimated $2.2 billion and $120 billion in agriculture and forestry respectively (Canadian Food Inspection Agency, 2008; Mogg, 2008).
Although DSV has negative impacts on the environment and economy, it is known to accumulate alkaloids. Alkaloids are naturally occurring organic nitrogen-containing bases which have important physiological effects on humans and other animals and are typically used for medicinal purposes. An example of a very well-known alkaloid used commonly in the medical field is morphine which is an opiate commonly used for pain relief.
Extremely little research has been performed with DSV and the development of a model system for investigating its chemistry and biochemistry is therefore be of interest. Understanding the metabolic pathways involved in producing the alkaloids in DSV could help in developing methods to control the plant or perhaps even ways to utilize it. To this end, cell calli from DSV may be potentially be used as a model system for studying the biosynthetic pathways involved in the production of the plant’s alkaloids.
Cell calli refers masses of undifferentiated cells in plants that grow in response to several environmental stimuli. In nature, calli often form as defense mechanism in response to wounding of the plant. The advantages of creating calli include presenting a simpler biological system and allowing easier manipulation of the growth environment. Of particular interest her is how calli allow for the formation of cell suspension culture which can be used as a bioreactor for large-scale production of metabolites and to study the biosynthetic pathways involved.
Growing calli involves first finding a proper medium for the callus. Culture medium is an artificial growth environment containing necessary nutrients for cell growth, and may contain sugars such as sucrose, several minerals (potassium, magnesium and others) and various hormones that affect the differentiation of cells to form callus or other tissue types. It provides a replacement for soil, the plants' natural growth medium. Hormones and other growth medium components can have major effects on the types and levels of alkaloids produced. They are therefore very beneficial for biochemical and pharmacological research purposes and are a key focus in various biotechnological research.
Materials and Methods:
The procedure for this investigation involved three main parts: (1) developing a suitable growth medium for calli cultivation, (2) surface-sterilizing explant material (tissue material from the plant of interest which is to be placed upon media), and (3) chemically analyzing the callus tissue utilizing an HPLC mass spectrometer. Once a suitable growth medium and growth conditions were identified, the callus was left grow for a period of 5 months with subculturing onto fresh medium every 3-4 weeks. The different stages of the callus growth are shown in Figure 3.
Figure 3 (a): Segments of DSV leaves and stems are placed on plates of Tobacco Media and Carrot Media
Figure 3 (b): DSV callus growth after 3 months in Carrot Media
Figure 3 (c): DSV callus growth after 4 months in Carrot Medium
Figure 3 (d): DSV callus growth after 5 months in Carrot Medium. The callus is ready to be chemically analyzed.
Cultivating and Finding a Suitable Growth Medium
The media used in this investigation were Tobacco Modified Callus Initiation Medium and Carrot Callus Initiation Basal Medium, both containing 3% sucrose and purchased from PhytoTech Labs. The hormones in the selected media included pre-added indole acetic acid (IAA)/Kinetin and 2, 4-dichlorophenoxyacetic acid (2,4-D), respectively. The media were sterilized at 121ºC for 30 minutes in an autoclave, and then both poured and left to solidify within 40-mm sterile petri dishes inside a HEPA filtered biosafety cabinet . The solidifying agent utilized instead of agar was Phytagel.
Surface-Sterilizing the Explants:
Since the explants utilized in this experiment were wild, this was an important step for minimizing the risk of bacterial/fungal infection from the wild plants to the calli cultures. However, it is important to note that this method proved to be ineffective against any bacteria or fungus residing within the explant which means that the plants were at risk of bacterial or fungal infection regardless of whether they are surface sterilized or not. The surface-sterilization procedure consisted of washing the explants in a series of solutions including tap water, distilled water containing 1-2 drops of 20% Tween, 70% ethanol, and 20% Chlorox © bleach solution. After the explant material was sterilized it was then placed onto the sterile medium and incubated at 25ºC with a 16-hr photoperiod (a time period in which the plant receives light).
Chemically Analyzing the Callus Tissue
The biochemical analysis of the calli took place at Vineland Research and Innovation Centre where the developed calli was analyzed for alkaloids by HPLC mass spectrometry. The method chosen to analyse the chemical properties of the callus was Electron Spray Ionisation (ESI). ESI uses a high voltage to ionize molecules to allow recognition by the detector. The alkaloid extraction procedure then was carried out as follows. The callus was frozen with liquid nitrogen and then ground into a fine powder using a mortar and pestle and placed into 1.5 mL microfuge tubes. The powder was dispersed in 1 mL of 75:25 methanol/distilled water solution and the tubes were vortexed (rapid oscillation of solution using vortex machine) until the powder was evenly suspended in the solution. The tubes were then sonicated (particles in the solution are agitated using soundwaves) and centrifuged at 14,000 rpm for 1 min to pellet the cells. The supernatant was then passed through a 0.2-µm syringe filter to get rid of any particles and placed into several Waters flasks ©. The flasks were then placed in the HPLC tray in a randomized order and the samples were analyzed.
Results:
While 40 explants were produced consisting of 50% leaves and 50% stems, a single leaf explant cultivated on carrot medium successfully generated DSV callus, representing a 2.5% success rate. This callus was continuously subcultured until eventually there were several calli which could tested. The growth conditions in which the calli were able to proliferate consisted of the carrot medium in a growth room where the calli were subjected to light exposure. To the best of our knowledge, this is the first ever protocol for developing callus from DSV. The HPLC mass spectrometer results showed that alkaloid contents appeared to be quite low in the callus compared to the quantities found in leaves of wild plants (Figure 4). Although numerous other alkaloids were observed to be produced in the callus, antofine was being accumulated in the highest concentration.
Figure 4: Chromatogram showing the concentration of the putative alkaloid antofine found within the seedlings compared to the amounts found within the callus. As can be seen in the graphs, the callus produced lower concentrations of the PIA antofine.
Figure 5: Chemical structure of antofine consisting of 3 aromatic rings and a bicyclic heterocycle along with 3 methoxy- groups. Antofine is the putative PIA that was found within both the seedling and callus samples in the highest concentrations among all of the alkaloids.
Discussion:
From this experiment, it was found that compact cell calli can be produced from Vincetoxicum rossicum leaves utilizing carrot basal medium and growing the plates with light exposure. It was also found that this plant species produces and accumulates phenanthroindolizidine alkaloids (PIAs) such as antofine, which are biologically active and possess antimicrobial, anti-inflammatory and anticancer properties (Chemler, 2009; Kempthorne, n.d.; Wang, 2014; Wang, 2012; Yang, 2005). Antofine’s structure consists of 3 aromatic rings and a bicyclic heterocycle along with 3 methoxy- groups as shown in Figure 5 making it a heteropentacyclic compound. It specifically has been tested and found to act as an antineoplastic agent that help in the prevention of tumour development (“Antofine,” n.d.). The medicinal properties of the PIAs found in DSV hold great potential and could prove to be revolutionary as calli cultures may develop faster than whole plants and allow for larger scale production due to their exponential growth capabilities. As well, the ability of callus to be produced form DSV will allow for the metabolic pathways involved in the production of PIAs to be studied. Since it was found that the callus produces antofine in lower concentrations than the natural plant, this suggests that variation in growth hormones and nutrients can affect the metabolic pathways in DSV.
Some of the antibacterial and antimicrobial traits could also potentially be implemented in the development of natural pesticides which could prove to be an economical alternative to the production of other pesticides. As well, understanding the biosynthesis of alkaloids in DSV and how this species is resistant to their own phytotoxin could bring about applications to control the invasiveness of this species. In summary, further research could lead to the discovery of medicinal, agricultural, and invasiveness controlling uses for this species. It could prove to be very beneficial for not only southern Ontario but perhaps even Canada or North America as a whole.
The findings in this project have opened a gateway into a vast array of research to be done with DSV. One such project could involve adding different types and quantities of hormones to the growth medium. After letting the calli grow in the new medium, mass spectrometry can be used to see if any biochemical pathways are affected which would be indicated by the presence of any new alkaloids or varying concentrations of previously identified alkaloids. Another project could be trying to create friable callus from the compact callus currently obtained in order to produce a cell suspension. In friable calli, the cells are loosely held together which allows the individual cells to separate much more easily. A cell suspension is another methodology for growing callus in which calli can grow at an exponential rate. The cell suspension involves the callus being placed in liquid media (same media without the phytagel) and being placed on a shaking plate. The shaking motion allows the individual cells to break off and separate in the liquid until the point where all the cells are separate. A proper cell suspension is much harder to obtain utilizing compact cell calli thus it is suggested to first grow friable calli and then utilize the friable calli within the cell suspension. As previously stated, friable calli allows for a suspension to form since the individual cells of the calli are loosely held together. Thereafter, the cells can be chemically analysed. A cell suspension could potentially allow for even further examination and understanding of the DSV biochemical pathways and lead to further possibilities of its use. With any possible discoveries of ways to integrate the alkaloids of this plant into pharmaceuticals, a cell suspension could also allow for the possibility of trying to maximize alkaloid production by upregulating the pathway involved.
Conclusion:
In conclusion, callus was able to be produced from the leaves of the Vincetoxicum rossicum plant. The growth conditions for the callus included utilizing Carrot Basal Growth Medium which employed 2, 4-dichlorophenoxyacetic acid (2,4-D) and several basic growth nutrients. The callus was subcultured several times in order to produce several calli for chemical testing. All calli were allowed to grow in a growth room with light exposure. Chemical testing for the callus was conducted at Vineland Research and Innovation Centre utilizing Electron Spray Ionisation (ESI) with an HPLC mass spectrometer. Through the testing it was found that the plant accumulates the alkaloid antofine which is of high interest to the pharmaceutical field as it has been found to possess antimicrobial, anti-inflammatory and anticancer properties.
Abbreviations:
DSV – Dog-Strangling Vine
PIA – Phenanthroindolizidine Alkaloids
HPLC – High Performance Liquid Chromatography
Acknowledgements
This project was performed at Brock University and Vineland Research and Innovation Centre as a part of the Brock University Mentorship Program. The study was carried out under the guidance of Dr. Vincenzo De Luca, Dr. David Liscombe, Ms. Christine Kempthorne, Ms. Alison Edge, and the De Luca Lab. Additional thanks for the efforts of Mme Julie Bedard, Mr. Peter Domarchuk, Mr. Lars Bruschke, and Sir Winston Churchill Secondary School.
References:
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Ishan Herath
Ishan Herath completed his high school education at Sir Winston Churchill Secondary School in St. Catharines, Ontario. He has consistently been an outstanding student, serving as an Arts Representative for his school’s student council and receiving numerous awards for both academics and extra curriculars including the Excellence in Education Award from the District School Board of Niagara and the Rotary Medalist Award for community service. Outside of academics, Ishan is an avid violinist playing in the Niagara Youth Orchestra for 8 years and 3 years as its concertmaster. Ishan now plays as part of the Brock University String Orchestra.