PRODUCTION OF PLANT DEFENSE COMPOUNDS IN CELL CULTURES AND THEIR EFFECTS ON BACTERIAL GROWTH

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INOM EXAMENSARBETE BIOTEKNIK, AVANCERAD NIVÅ, 30 HP , STOCKHOLM SVERIGE 2016

PRODUCTION OF PLANT

DEFENSE COMPOUNDS IN CELL

CULTURES AND THEIR EFFECTS

ON BACTERIAL GROWTH

JUNE WINBLAD

KTH

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PRODUCTION OF PLANT DEFENSE

COMPOUNDS IN CELL CULTURES AND

THEIR EFFECTS ON BACTERIAL GROWTH

Master degree thesis report

School of Biotechnology, KTH, Stockholm

Supervisors:

Docent Anna Ohlsson

Docent Gunaratna Kuttuva Rajarao

Examiner:

Professor Pål Nyrén

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Abstract

The textile industry is using antimicrobial substances to prevent the growth of

microorganisms during storage and transport. Most antimicrobials in use are synthetic chemical compounds, many of which have proved to be toxic to human health and to the environment. Some of these substances are not allowed to be used as biocides in the EU. A prerequisite for an effective ban is the availability of feasible alternatives. One possible solution would be to identify safe antimicrobial agents from nature.

The purpose of this study was to treat plant cell cultures with elicitors (defense inducing substances or preparations) and natural stress signal compounds in order to increase their production of antimicrobial substances. A shoot differentiated Pisum sativum (garden pea) culture and an undifferentiated fine suspension Populus trichocarpa (black cottonwood) culture were selected for the experiments. Solutions of the stress signalling compounds nicotinamide, nicotinic acid, salicylic acid and methyl jasmonate and the elicitor chitosan were used to induce defense metabolism. Phenolic compounds constitute a group of

secondary metabolites in plants, which are utilized as markers of an activated defense. Cell extracts from control (untreated) and treated cultures are tested for their effects on bacterial growth.

The amount of total phenolic substances was analyzed in cell material as well as in culture medium after harvest of the cultures. The largest effect was a 7.5 times increase in phenolic content in cell material after methyl jasmonate treatment. Salicylic acid caused increased phenolic content in Populus culture medium, but overall the excretion of phenolic substances to the culture medium was not influenced to any great extent. The major part of the phenolics was retained in the cell material.

Extracts from untreated Populus cells proved to have antibacterial properties against Bacillus

subtilis in an agar plate assay. The effect was even stronger with extracts from salicylic acid

treated culture.

Chitosan did not affect production of phenolic compounds in Populus cells but extracts from the same cells had antibacterial properties when B. subtilis was grown both on solid (agar) and in liquid medium, when compared to extract from untreated culture. Also extract from cells treated with a combination of nicotinamide and methyl jasmonate had an inhibiting effect in liquid medium, while nicotinamide alone had the opposite effect, leading to stimulated growth of B. subtilis.

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Sammanfattning

Textilindustrin använder antimikrobiella ämnen för att förhindra tillväxt av mikroorganismer under lagring och transport. De flesta antimikrobiella medel som används är syntetiska

kemiska föreningar varav många har visat sig vara giftiga för människors hälsa och för miljön. Några av dessa ämnen är inte tillåtna som biocider inom EU. En förutsättning för effektiva förbud är tillgången på genomförbara alternativ. En möjlig lösning skulle vara att identifiera icke toxiska antimikrobiella ämnen från naturen.

Syftet med den här studien är att behandla växtcellsodlingar med elicitorer (försvars-inducerande substanser och preparationer) och naturliga stressignalsubstanser för att öka cellernas produktion av antimikrobiella ämnen. En skott-differentierad kultur av Pisum

sativum (trädgårdsärt) och en odifferentierad finsuspensionskultur av Populus trichocarpa

(jättepoppel) valdes ut för experimenten. Lösningar av stressignalsubstanserna nikotinamid, nikotinsyra, salicylsyra och metyljasmonat samt elicitorn kitosan användes för att påverka försvarsmetabolismen. Fenolföreningar är en grupp sekundärmetaboliter i växter som utnyttjas som markörer för ett aktiverat försvar. Cellextrakt från obehandlade respektive behandlade kulturer testades avseende effekt på bakteriertillväxt.

Mängden totala fenoliska substanser analyserades i cellmaterialet och i odlingsmediet efter skörd av kulturerna. Den största effekten var en 7,5 gånger ökad halt av fenoliska substanser i cellmaterialet efter behandling med metyljasmonat. Salicylsyra orsakade ökad halt av totala fenoler i Populus odlingsmedium, men totalt sett påverkades inte utsöndringen av fenoliska substanser till odlingsmediet i någon större utsträckning. Den största andelen fanns kvar i cellmaterialet.

Extrakt från obehandlade Populus-celler visade sig ha antibakteriell effekt mot Bacillus

subtilis odlad på agarplatta. Effekten var ännu starkare med extrakt från kulturer behandlade

med salicylsyra.

Kitosan påverkade inte produktionen av fenoliska föreningar i Populus-celler, men extrakt från samma celler hade antibakteriella egenskaper vid odling av B. subtilis både på fast medium (agar) och i flytande medium, vid jämförelse med odlingsextrakt från obehandlad kultur. Även extrakt från celler behandlade med en kombination av nikotinamid och

metyljasmonat hade en hämmande effekt i flytande medium, medan nikotinamid ensamt hade motsatt effekt, nämligen stimulerad tillväxt av B.subtilis.

Från den här studien finns inga direkta bevis för att fenoliska föreningar är ansvariga för de antibakteriella egenskaperna hos Populus-kulturen. Men resultaten pekar på en möjlighet att utnyttja växter eller växtcellskulturer för framställning av ämnen med antibakteriell aktivitet för användning i olika tillämpningar.

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List of abbreviations

dH2O: deionized water

IZ: inhibitory zone. That is the area on an agar plate where there is no bacterial growth because a plant extract is placed.

MBC: minimum bactericidal concentration. That is the lowest concentration of plant extract resulting in killing ≥ 99.9% of an original inoculum of microorganisms.

MeJA: methyl jasmonate MeOH: methanol

MIC: minimum inhibitory concentration. That is the lowest concentration of plant extract resulting in visible inhibition of microorganism growth after overnight incubation. NIA: nicotinic acid

NIC: nicotinamide OD: optical density

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1. Introduction

1.1 Problem

The textile industry is often using antimicrobial substances to prevent the growth of microorganisms during storage and transportation. Today a significant part of textiles and ready-made clothes sold in Europe and worldwide are manufactured in the warm and humid areas of East and Southeast Asia. Most antimicrobials in use today are synthetic chemical compounds, such as silver compounds, triclosan and triclocarban, many of which have proved to be toxic to human health as well as to the environment. These compounds are spread via the food chain, and often not degraded. They may cause cell death and endocrine disruption, affect reproduction, pollute water and soil, and damage aquatic organisms. (KEMI 2012, 2013; Bergman et al. 2012)

To counter the application of toxic antimicrobials, EU has issued strict regulations. Some chemical antibacterial substances are no longer allowed to be used as biocides in the EU. The Swedish government in 2013 introduced a government bill “Towards a non-toxic everyday life”, to strengthen the implementation of the regulations. (ECHA 2012; Regeringskansliet 2013; Mossialos et al. 2010).

Implementation of political decisions requires technically feasibly solutions. The textile

industry therefore needs access to safe antimicrobial agents to replace the toxic chemical ones. Otherwise these regulations would not be implemented or accepted by the industry. One possible solution would be to develop safe antimicrobial agents from nature.

1.2 Literature review

Naturally safe antibacterial substances from plants have been used since ancient times, especially as medicine. Some natural substances from plants are toxic to insects and microorganisms but not necessarily to humans. Some of this ancient knowledge has been ignored in industrialized societies. The reasons are that synthetic antimicrobials often are very effective and can be produced at low cost. The danger of these chemicals to human health and the environment indicates the necessity to identify nontoxic natural substances to replace the toxic chemicals in use today.

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1.2.1 Effects against pathogenic microorganisms

Extracts of some Mediterranean plants, Olea europaea, mastic gum, and Inula viscosa, showed excellent antibacterial properties against bacteria causing oral diseases (Karygianni et

al. 2014).

Jadhav et al. (2014) found that extract of some Indian herbs, Terminalia paniculata,

Terminalia crenulata Roth, Cuscuta reflexa Roxb, Bridelia retusa Spreng and Syzygium cumini Linn, had inhibitory effects against sexually transmitted pathogens, Neisseria gonorrhoeae, Haemophilus ducreyi and Candida albicans.

Some plant essential oils and compounds had antibacterial effects against Escherichia coli O157:H7 and Salmonella enterica in apple juice. (Friedman et al. 2004). Some of the most effective essential oils and compounds which were active against both bacteria species were oregano oil, cinnamon leaf oil, lemongrass oil, lemon oil, carvacrol, geraniol, eugenol and citral.

Essential oils from the Turkish plants Satureja spicigera and Thymus fallax have shown

excellent antibacterial effects against 25 plant pathogenic bacteria strains and seed infections (Kotan R. et al. 2010). The pathogens’ host plants include cotton, apple, pear, apricot, tomato, lettuce, cabbage, radish, pepper, zinnia and geranium.

Both antioxidant and antimicrobial effects against aerobic bacteria and mould for lamb patties preservation in Egypt were reported for extracts from four natural plants, ginseng, jatropha, jojoba and ginger (Ibrahim et al. 2011).

It is important to realize that even though some plant extracts show antibacterial effects, the same extracts may also have either antagonistic or synergistic effects together with

conventional antibiotics tested against E. coli causing human infections. (Ushimaru et al. 2012).

While this report was being written, the Nobel Committee awarded half of the 2015 Nobel Prize in Physiology or Medicine to the Chinese researcher Tu Youyou. She rediscovered and purified the effective compound, Artemisinin, from the herb Artemisia annua (sweet

wormwood) to cure malaria. Her discovery shows that there is an effective plant-based solution when synthesized drugs failed due to development of drug resistance in the malaria parasite.

1.2.2 Effects against Gram-positive bacteria

Many plant extracts have showed inhibitory effects against Gram-positive bacteria.Nostro et

al. (2012) found that extracts of the natural plant or the cell culture of Limonium avei, an

endangered herbal species from the central Mediterranean coastal region, have showed good inhibitory effect against Gram-positive bacteria, Staphylococcus aureus, Staphylococcus

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methicillin resistant and sensitive strain S. aureus. The natural plant extract has better effects than those of the cultured plant cells.

Gonçalves et al. (2009) found that the extract of the Mediterranean plant Drosophyllum

lusitanicum showed strong antimicrobial effects against S. epidermidis, S. aureus, Streptococcus pyogenes and Enterococcus faecalis.

Extracts from two Australian plants, Eremophila neglecta (Ndi et al. 2007) and Eremophila

microthec (Barnes et al. 2013), showed antibacterial effects against S. aureus, S. pyogenes

and Streptococcus pneumoniae.

Tussilago farfara and Urtica dioica are both found in Europe and other parts of the world and

have since ancient times been used in traditional medicine. T. farfara has for example been used to treat cough and U. dioica for treatment of a range of diseases. Turker et al. (2008) found that leaf extracts from these plants, as well as extracts from Helichyrsum plicatum flowers and from Solanum dulcamara aerial parts showed strong inhibitory effects on S.

pyogenes, S. aureus and S. epidermidis.

Joray et al. (2011) reported that the plant-derived compounds,

23-methyl-6-O-desmethylauricepyrone and (Z,Z)-5-(trideca-4,7-dienyl) resorcinol, had effects against resistant S. aureus. The synergistic effect of desmethylauricepyrone with erythromycin or gentamicin had decreased the usage of the two antibiotics by 300 and 260 times respectively.

1.2.3 Effects against both Gram-positive and Gram-negative bacteria

Most reports regarding effects of plant extracts against Gram-negative bacteria also include Gram-positive bacteria. However, Chang et al. (2008) reported that the essential oil of

Cinnamomum osmophloeum leaf has shown good inhibitory effects against Gram-negative

bacteria Legionella pneumophila, without including any Gram-positive bacteria in their investigation. The researchers also found that cinnamaldehyde was the dominate component (>90%) in the essential oil and was the important compound responsible for the antibacterial properties.

Bartfay et al. (2012) found that extract of the whole herb Epilobium angustifolium showed excellent antibacterial properties, with great statistical significance, against a variety of both Gram-positive and Gram-negative bacteria, for instance Micrococcus luteus, S. aureus, E. coli and Pseudomonas aeruginosa. Moreover, the inhibitory effects of the extract in culture have shown better results than those of vancomycin and tetracycline.

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The plant Eremophila neglecta had good effects against a long row of Gram-positive bacteria, such as S. pneumonia, S. pyogenes, B. subtilis, Bacillus cereus, Enterococcus faecium, E.

faecalis, Mycobacterium fortuitum, Mycobacterium chelonae, and Erysipelothrix

rhusiopathiae, and also the Gram-negative bacterium Moraxella catarrhalis (Anakok et al.

2011).

It has been reported that both the shape and the membrane surface of bacteria S. aureus and E.

coli were damaged after the bacteria were treated with extracts of the medicinal plant

Melastoma candidum (Yong et al. 2014).

Interesting for the present investigation is the production of terpenoid and phenolic

compounds studied by Lv et al. (2012). It was reported that the essential oil of the Chinese plant Chimonanthus praecox showed antioxidant effects and excellent inhibitory effects against Gram-positive bacteria B. subtilis and Gram-negative bacterial Salmonella typhi. The researchers identified that the main compounds in the essential oil were sesquiterpenoids and their derivatives (35.97%), monoterpenes and their derivatives (15.87%) and phenolic

compounds (0.35%). The authors suggest that the synergistic action of the three types of compounds contributes to the antibacterial effect of the essential oil.

A study with relevance for the long term goal of the present investigation is the report by Baliarsingh et al. (2013) in which they show that the extract of Mangifera indica leaves from plants grown in India gave good antibacterial effects against both positive and Gram-negative bacterial strains, S. aureus, S. pyogenes, E. coli and Klebsiellapneumoniae and when

the extract was used to dye silk and cotton yarns with cationic surfactant, the colors were strengthened and bacterial growth in the yarns was inhibited.

1.2.4 Effects against fungi

Some plant extracts have inhibitory effects against fungi. Gonçalves et al. (2009) found that the extract of Drosophyllum lusitanicum had not only effects against Gram-positive bacteria as mentioned earlier but also against the fungi, C. albicans, Candida famata, Candida

catenulata, Candida guilliermondi, Yarrowia lipolytica, Trichosporon mucoides, Trichosporon beigelii, and Cryptococcus neoformans.

Extracts of the plants Tagetes minuta, Lippia javanica, Vigna unguiculata and Amaranthus

spinosus grown in South Africa were reported to have inhibitory effects against the

mycotoxigenic fungi Fusarium verticillioides and Fusarium proliferatum (Thembo et al. 2010).

In Nepal, Paudel et al. (2014) found that the essential oil of a parasitic vine, Cuscuta reflexa Roxb., had moderate inhibitory effect against Aspergillus niger.

The vegetable-pathogenic fungus Colletotrichum lagenarium can cause anthracnose of

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that extracts of Cinnamomum camphora (L.) has excellent inhibitory effect against this

fungus and that the fungus hyphae became shortened after the treatment with the extracts.

1.3 Plant cell culture

1.3.1 Induction of plant defense response

In some studies mentioned in Section 1.2 the researchers have tried to identify the effective compounds in plant extracts responsible for the antimicrobial activities. These compounds are different kinds of secondary metabolites in plant cells. Such compounds can be produced and released when plants defend themselves against biotic or abiotic stresses in nature. Such secondary metabolites can be toxic to insects and microorganism but not necessarily to humans. (Berglund et al. 2015)

Phenolic compounds are one class of secondary metabolites in plants (Cheynier 2012) and are well-studied as markers of plant defense system (Ruiz-García and Gómez-Plaza 2013). They have also been found to have antimicrobial properties (Puupponen-Pimiä et al. 2001; Alves et

al. 2013). Moreover, phenolic compounds can be easily analyzed and compared with results

of other researchers as standard methods are available (Swain and Hillis 1959). In this project the production of total phenolic compounds was thus studied as an indicator of plant defense response.

The production of phenolic compounds and other secondary metabolites can be induced by exposure of plants or plant cells to chemical elicitors or stress signaling compounds (Berglund

et al. 2015). In the present study, the stress signalling compounds nicotinamide (NIC), nicotinic acid (NIA), salicylic acid (SA) and methyl jasmonate (MeJA) and the elicitor chitosan have been added to plant cell cultures with the purpose to increase the production of antimicrobial substances.

NIC and NIA, as the common forms of Vitamin B3, have been found to be able to induce defense-related metabolism as stress signaling compounds for defense response in plant cells (Berglund et al. 1993a, 1993b, 2015; Berglund and Ohlsson 1995; Louw et al. 2000; Ohlsson

et al. 2008). NIC has also been suggested to have priming effect, that is, NIC may strengthen

a second stress-signal and thus result in a higher level of defense response (Berglund et al. 2015).

SA itself, originally purified from the bark extract of willow tree, has for years been known for its anti-inflammatory effect in humans. SA is also an important plant hormone in

regulating the defense response in plants against microbial pathogens (Kumar 2014). When plant cells were induced with SA it resulted in the accumulation of reactive oxygen species and expression of defense genes (Vasyukova and Ozeretskovskaya 2007). SA has been shown to stimulate phenolic synthesis in Salvia miltiorrhiza cell culture (Dong et al. 2010) and in peas combined with a low pH treatment (McCue et al. 2000).

As an elicitor, chitosan stimulated plumbagin formation in the cell cultures of Plumbago

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al. 2002). Chitosan has been found to stimulate the defense response of plants by inducing

accumulation of phytoalexins (Hadrami et al. 2010). Chitosan has also showed the effect of increasing total polyphenols in grapes and strawberries (Ruiz-García and Gómez-Plaza 2013). MeJA has been demonstrated to effectively induce lipid peroxidation and lipoxygenase and to play an important role in signaling and activating defense responses in tobacco cells (Dubery

et al. 2000). MeJA has also been found to enhance plant defense response after subsequent

stress (Delaunois et al. 2014). MeJA is a derivative from jasmonic acid (JA) and has similar activity as JA in increasing the biosynthesis of polyphenols in plants (Ruiz-García and Gómez-Plaza 2013). JA has showed the eliciting effect of increasing production of phenylpropanoids and naphtodianthrones in Hypericum perforatum L. cell culture (Gadzovska et al. 2007).

NIC, NIA, SA, chitosan and MeJA, were therefore used in this project with the purpose to induce plant defense systems and to stimulate production of antimicrobial substances in plant cell cultures.

1.3.2 Treatment of plant cell cultures

In the studies mentioned in Section 1.2, it can be seen that the researchers often choose to study the common plant species in their regions. In this degree project the species Pisum

sativum (garden pea) and Populus trichocarpa (black cottonwood) were used for studies as

representatives as they are common plant species in many parts of the world. Populus belong to Salicaceae Family, also called as Willow Family.

Extract of pea peel has been found to have antioxidant and antimicrobial effects (Hadrich et al. 2014). Extract of pea seed has been used to control biofilm formation by inhibiting P.

aeruginosa and the tannins and phenolic compounds were found to be responsible for its

antibacterial property (Dazal et al. 2015). This leads to a suggestion of using pea peel waste for production of antibacterial compounds. Antimicrobial flavonoids were found from the twigs of Populus and effective against plant pathogens, Pseudomonas lachrymans, Ralstonia

solanacearum, Xanthomonas vesicatoria and Magnaporthe oryzae. (Zhong et al. 2012).

From the buds of Populus anitibacterial dihydrochalcone derivatives were found to be effective against S. aureus (Lavoie et al. 2013).

Cell cultures of these two plants (P. sativum and Populus) have been well-studied for their defense mechanisms by a research group at the School of Biotechnology, KTH. This degree project was the first attempt to investigate the production levels of phenolic compounds by the two plant cell cultures after treatment with NIC, NIA, SA, chitosan and MeJA, as well as resulting effects on bacterial growth. The experiments were carried out with simple combinations to obtain experience and initial screening.

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activities, more additions (NIC, SA, chitosan and MeJA) were made to the Populus culture. The suggested priming effects of NIC were also investigated.

According to findings from previous research in plant defense mechanisms at KTH the plant cells need some time, in this case three days, to perform the defense response after being stressed. Some defense reactions may be missed if it takes a longer time before the cells are harvested. NIC as a priming factor also needs time, in this case one day, to “prime” the cells so that they could become more sensitive to stress factors. In this degree project the rest of the additions to the cell cultures were therefore done three days before harvesting and NIC as a priming factor was added one day earlier.

The technology of plant cell cultivation was used in this project. The advantage is that the researcher can obtain a simplified and controlled experimental system and get quick results within a short period (Berglund et al. 1993; Ohlsson et al. 2006). Once the potential

compounds are identified from the cells it is possible to produce them in large scale under controlled conditions. In this way the compounds can be produced without modifying their structure and functionality. The disadvantage is that the result obtained from plant cell cultures may not in all cases reflect the effects of natural plants (Nostro et al. 2012).

1.3.3 To investigate effects on bacterial growth

Gram-positive bacteria B. subtilis and Gram-negative E. coli were used in the project. The two bacteria are well-studied and often used as representatives of the two Gram strains. They are also relatively safe when being used in a non-clinic laboratory.

To investigate bacterial growth, agar plate assay and microtiter (96-well) plate assay were used in this project. The advantage of agar plate assay is that it is simple and quick way to obtain qualitative results. A disadvantage is that one doesn’t know what might have happened during incubation if, for instance, the bacteria were inhibited first but then came back to take over. The advantage of microtiter plate assay is, however, that the dynamic process can be observed by recording the growth curve of the microorganisms, and that it can provide

quantitative results. The disadvantage is that it is more complicated and very much dependent on accurate and sensitive equipment.

1.4 Aims and Goals

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2. Materials and methods

The experiments performed in the degree project consist of three major parts: 1) to treat plant cell cultures with elicitors and stress signalling compounds, 2) to extract secondary

metabolites from the cell material and to analyze the production of phenolic compounds from the extracts, and 3) to investigate the growth of bacteria when they are exposed to the extracts.

2.1 Treatment of cell cultures

2.1.1 Plant material

Pisum sativum shoot (PS-S) tissue differentiated culture and Populus trichocarpa (Populus)

fine suspension culture were available in the laboratory at the School of Biotechnology, KTH. The PS-S culture was cultivated in modified Murashige-Skoog (MS) medium MS-III GA3 (for formula of the medium see Appendix 1), and sub-cultivated every second week for maintenance (Berglund et al. 1993b).

The Populus cell culture was cultivated in modified MS-I medium (for formula of the medium see Appendix 1), and sub-cultivated weekly for maintenance in the same way as described for hybrid aspen suspension culture (Ohlsson et al. 2006).

Both cultures were grown at room temperature at a photoperiod of 12 h fluorescent light and 12 h dark. All work with plant cultures until the point of harvest were carried out under sterile conditions.

2.1.2 Chemicals

The compounds used were NIC (Merck), NIA (Merck), SA (Analar), chitosan (Aldrich) or MeJA (Aldrich). The preparation of their stock solutions is specified in Table 1. Chitosan was solubilized in 0.1 mM HCl at 65°C for a short time, whereafter pH was adjusted to

approximate 5 with 1 M NaOH. The chitosan stock solution was sterilized by autoclaving. The other stock solutions were filter-sterilized with 0.20 µm non-pyrogenic sterile filters.

Table 1 Preparation of stock solutions.

Compound Weight

(g)

Solvent Stock concentration

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2.1.3 Treatments of plant cell cultures

Treatment 1

1. Preparing 12 sterilized erlenmeyer flasks of modified MS-III GA3 medium, each containing 50 ml.

2. Selecting a flask of well-grown PS-S cell culture from previous sub-cultivations. Filtering the culture to get the shoot tissues, carefully dividing the tissues into tiny pieces, weighing and inoculating 2.5 g (wet weight) of fresh parts into each of the 12 flasks.

3. Incubating the 12 flasks of cultures for 14 days. On the 11th day, adding NIC, NIA or SA into the cultures in triplicates. The additions are specified and the cultures are coded in Table 2.

4. Harvesting the tissue cultures on the 14th day.

Table 2 Additions to cell cultures and codes given to the cultures in Treatment 1.

Culture no. Addition Final concentration

(mM) Code* 1 5 ml dH2O - 1-PS-untr 2 5 ml dH2O - 3 5 ml dH2O - 4 5 ml 20 mM NIC 2 1-PS-NIC 5 5 ml 20 mM NIC 2 6 5 ml 20 mM NIC 2 7 5 ml 20 mM NIA 2 1-PS-NIA 8 5 ml 20 mM NIA 2 9 5 ml 20 mM NIA 2 10 5 ml 10 mM SA 1 1-PS-SA 11 5 ml 10 mM SA 1 12 5 ml 10 mM SA 1

(* the code consists of three parts: the first part, ‘1’, means it is from Treatment 1. The second part, ‘PS’, means PS-S culture. The third part indicates which substance was used. In the third part ‘untr’ means untreated, that is with no addition.)

Treatment 2

1. Preparing 12 sterilized erlenmeyer flasks of modified MS-I medium, each containing 50 ml.

2. Selecting a flask of well-grown Populus cell culture from previous sub-cultivations. Filtering the culture to get the cells, inoculating 2.5 g (wet weight) of cells into each of the 12 flasks.

3. Incubating the 12 flasks of cultures for 7 days. On the 4th day, adding SA, chitosan or MeJA into the cultures in triplicates. The additions are specified and the cultures are coded in Table 3.

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Table 3 Additions to cell cultures and codes given to the cultures in Treatment 2.

Culture no. Addition Final concentration

(mM) Code* 1 5 ml dH2O - 2-PO-untr 2 5 ml dH2O - 3 5 ml dH2O - 4 5 ml 10 mM SA 1 2-PO-SA 5 5 ml 10 mM SA 1 6 5 ml 10 mM SA 1 7 0.75 ml 6.7 mg/ml Chitosan 0.1 mg/ml 2-PO-chi 8 0.75 ml 6.7 mg/ml Chitosan 0.1 mg/ml 9 0.75 ml 6.7 mg/ml Chitosan 0.1 mg/ml 10 0.2 ml 50 mM MeJA 0.2 2-PO-MeJA 11 0.2 ml 50 mM MeJA 0.2 12 0.2 ml 50 mM MeJA 0.2

(* the code consists of three parts: the first part, ‘2’, means it is from Treatment 2. The second part, ‘PO’, means Populus culture. The third part indicates which substance was used. In the third part ‘untr’ means untreated, that is with no addition, and ‘chi’ means chitosan.)

Treatment 3

1. Preparing 15 sterilized erlenmeyer flasks of modified MS-I medium, each containing 50 ml.

2. Selecting a flask of well-grown Populus cell culture from previous sub-cultivations. Filtering the culture to get the cells, inoculating 2.5 g (wet weight) of cells into each of the 15 flasks.

3. Incubating the 15 flasks of culture for 7 days. On the 3rd day, adding NIC as a priming factor into 8 of the 15 flasks of culture. On the 4th day, adding SA, chitosan or MeJA into the cultures in duplicates. The additions are specified and the cultures are coded in Table 4.

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Table 4 Additions to cell cultures and code of cultures in Treatment 3.

Culture

no. Addition on the 3rd day concentration Final (mM)

Addition on the

4th day concentration Final (mM) Code* 1 0.5 ml dH2O - - 3-PO-untr 2 0.5 ml dH2O - - 3 0.5 ml dH2O - - 4 0.5 ml 200 mM NIC 2 - 3-PO-NIC 5 0.5 ml 200 mM NIC 2 - 6 0.5 ml dH2O - 2 ml 25 mM SA 1 3-PO-SA 7 0.5 ml dH2O - 2 ml 25 mM SA 1 8 0.5 ml dH2O - 0.75 ml 1 mg/ml chitosan 0.015 mg/ml 3-PO-chi 9 0.5 ml dH2O - 0.75 ml 1 mg/ml chitosan 0.015 mg/ml 10 0.5 ml 200 mM NIC 2 2 ml 25 mM SA 1 3-PO-NIC+SA 11 0.5 ml 200 mM NIC 2 2 ml 25 mM SA 1 12 0.5 ml 200 mM NIC 2 0.75 ml 1 mg/ml chitosan 0.015 mg/ml 3-PO-NIC+chi 13 0.5 ml 200 mM NIC 2 0.75 ml 1 mg/ml chitosan 0.015 mg/ml 14 0.5 ml 200 mM NIC 2 0.2 ml 50 mM MeJA 0.2 3-PO-NIC+MeJA 15 0.5 ml 200 mM NIC 2 0.2 ml 50 mM MeJA 0.2

(* the code consists of three parts: the first part, ‘3’, means it is from Treatment 3. The second part, ‘PO’, means Populus culture. The third part indicates which substance(s) were used. In the third part ‘untr’ means untreated, that is with no addition, and ‘chi’ means chitosan.)

2.1.4 Harvesting of cultures

Plant cell cultures were harvested by vacuum filtration. The mass weight was measured. The plant cell material was collected into Falcon tubes and quickly frozen in liquid nitrogen. The tubes were then stored in a refrigerator at - 18 oC for later analysis.

The pH of the filtered medium was also measured.

2.2 Extraction of secondary metabolites and analysis of phenolic

production

2.2.1 Extraction of secondary metabolites from plant cell material

The harvested cell material was first homogenized in liquid nitrogen and then extracted with 80% MeOH as follows (Swain and Hillis 1959):

1. Place 100 mg of the homogenized cell material in a 1.5 ml Eppendorf tube. 2. Add 1 ml 80% MeOH into the tube, and vortex the mixture thoroughly for ca 2

minutes.

3. Centrifuge the mixture at 13 000 rpm for 10 minutes at room temperature. 4. The supernatant is the extract and is transferred into a new Eppendorf tube. 5. The concentration of the extract is 0.1 mg/µl. (The concentration is defined as the

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When a large amount of cell material, for instance 5 g, was extracted the procedure was as follows:

1. Place 5 g of cell material into a 50 ml Falcon tube.

2. Add 20 ml 80% MeOH into the tube, and vortex the mixture thoroughly for ca 2 minutes.

3. Centrifuge the mixture at 4750 rpm for 20 minutes at 4 oC. 4. Transfer the supernatant into a new 50 ml Falcon tube.

5. Add 20 ml 80% MeOH into the sediment, vortex thoroughly for ca 2 minutes. 6. Centrifuge the secondary mixture at 4750 rpm for 20 minutes at 4 oC.

7. Transfer the supernatant again into the same Falcon tube as in step 4.

8. Add another 10 ml 80% MeOH into the sediment again, vortex thoroughly for ca 2 minutes.

9. Centrifuge the third-time mixture at 4750 rpm for 20 minutes at 4 oC. 10. Transfer the supernatant again into the same Falcon tube as in step 4 and 7. 11. The supernatant is the extract. The concentration of the extract is 0.1 mg/µl.

When the primary extract had to be concentrated, a rotative evaporation method was used to concentrate the extract at 150 mbar for 25-30 minutes at 40 oC. The volume could be reduced from 50 ml to about 10 ml.

2.2.2 Analysis of production of phenolic compounds from cell material

Phenolic compounds were analyzed according to the Swain and Hillis method (1959). A standard curve was created each time when samples were analyzed as follows:

1. For a standard curve, take a series of volumes such as 0, 10, 25, 50, 100 and 200 µl, respectively, of 0.1 mg/ml chlorogenic acid into a 1.5 ml Eppendorf tube. For cell extract samples, take a suitable volume.

2. Add dH2O to a total volume of 700 µl in each tube.

3. Add 50 µl Folin-Denis reagent into each tube, mix and then let it stand still for 3 minutes for reactions.

4. Add 100 µl saturated Na2CO3 and mix.

5. Add 150 µl dH2O and mix, then let it stand still for 1 hour for reactions. 6. Centrifuge the mixtures at 13 000 rpm for 10 minutes.

7. Transfer 700 - 800 µl the supernatant into a cuvette. 8. Measure absorbance at 765 nm.

2.3 Antibacterial assay

Antibacterial assay in this project included agar plate assay and microtiter (96-well) plate assay.

2.3.1 Bacterial strains

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Both bacteria were grown with Nutrient Agar medium. B. subtilis was incubated overnight at 30 oC while E.coli was incubated overnight at 37 oC. Two agar plates of each bacterial strain were kept at 4 oC and subcultured every 2 weeks for maintenance.

2.3.2 Agar plate assay

Both bacterial strains were tested with the agar plate method. The procedure was as follows: 1. Autoclaved Nutrient agar was poured to each petri plate to a depth of 4 mm,

equivalent of 25 ml liquid agar to a 100-mm plate. The agar plates were left to solidify until there was no visible liquid on the surface.

2. Take the bacterial overnight culture, dilute the culture with sterile saline solution to an OD of 0.1 at 600 nm.

3. Spread 150 µl the diluted culture evenly on each Nutrient Agar plate. Leave the culture to soak into the agar.

4. Use a sterile metal cylinder to punch 4 wells with a diameter of 6.5 mm in each agar plate. The wells should be placed evenly on the agar surface as shown in Figure 1. 5. Add 30 µl solvent into a well as negative control, load cell extract samples with a

series of 3 doses into the other 3 wells. The load with lower dose was diluted with dH2O. One agar plate for each sample of the cell extracts. The load of samples is illustrated in Figure 1. The doses added for each agar plate assay are specified in Table 5.

6. Incubate the agar plates overnight.

7. Observe the agar plate next morning if there is any inhibitory zone(s). Inhibitory zone (IZ) is the zone where there is no bacterial growth around the well. The larger the zone is, the better the inhibitory effect against bacteria is. IZ examples are shown in Figure 1.

8. Use a ruler to measure the diameter of IZ(s) if it appears. When measuring, round up to the next millimeter including the diameter of the well.

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Table 5 Doses of cell extracts used in Agar plate assay and bacteria tested.

Samples Concentration

(mg/µl) 30 µl 20 µl 10 µl Dose in different load (mg) bacteria Tested 1-PS-untr 1-PS-SA 0.1 -"- 3 -"- 2 -"- 1 -"- B.subtilis 1-PS-untr 1-PS-NIC 1-PS-NIA 1-PS-SA 0.25 -"- -"- -"- 7.5 -"- -"- -"- 5 -"- -"- -"- 2.5 -"- -"- -"- B.subtilis 2-PO-untr 2-PO-MeJA 0.1 -"- 3 -"- 2 -"- 1 -"- B.subtilis 2-PO-untr 2-PO-SA 2-PO-chi 2-PO-MeJA 0.25 -"- -"- -"- 7.5 -"- -"- -"- 5 -"- -"- -"- 2.5 -"- -"- -"- B.subtilis E.coli 3-PO-untr 3-PO-NIC 3-PO-SA 3-PO-chi 3-PO-NIC+SA 3-PO-NIC+chi 3-PO-NIC-MeJA 0.4 -"- -"- -"- -"- -"- -"- 12 -"- -"- -"- -"- -"- -"- 8 -"- -"- -"- -"- -"- -"- 4 -"- -"- -"- -"- -"- -"- B.subtilis

2.3.3 Microtiter 96-well plate assay

Populus cell extracts from Treatment 3 were tested against B. subtilis with the 96-well plate

method. The procedure was as follows:

1. Dilute the Nutrient Broth medium with sterile dH2O to 1:10.

2. Take the bacterial overnight culture, dilute it with 1:10 medium to an optical density (OD) of 0.1 absorbance at 600 nm.

3. Add the cell extract to a sterile microtiter 96-well plate (Sarstedt) (see the example design in Figure 2), the total volume of each well was 100 µl, as detailed below: 1:10 medium blank: 100 µl.

Culture control (OD=0.1): 100 µl.

Sample, dose 1: 5 µl cell extract + 5 µl dH2O + 90 µl culture. Sample, dose 2: 10 µl cell extract + 90 µl culture.

Solvent control: 10 µl solvent + 90 µl culture.

The cell extracts from Treatment 3 had a concentration of 5 mg/µl. The doses applied were 25 or 50 mg.

4. Add 100 µl dH2O into each of the remaining wells to keep moisture in the plate, 5. Incubate the plate with lid overnight in a FLUOstar Optima device (BMG Labteck) at

30 oC with orbital shaking.

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Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7

1 2 3 4 5 6 7 8 9 10 11 12 A dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O B dH2O Medium Blank Culture control

Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Solvent control dH2O C dH2O Medium Blank Culture control

Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Solvent control dH2O D dH2O Medium Blank Culture control

Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Solvent control dH2O E dH2O Medium Blank Culture control

Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Solvent control dH2O F dH2O Medium Blank Culture control

Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Solvent control dH2O G dH2O Medium Blank Culture control

Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Solvent control

dH2O

H dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O

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3. Results

This chapter presents the results from 1) measurement of cell mass and pH in plant cell culture medium, 2) analysis of content of phenolic compounds in the cell extracts and in plant cell culture medium and 3) investigation of the effects of the cell extracts on bacterial growth.

3.1 Cell mass and pH

In treatment 1, all additions to the PS-S culture caused a small decrease in growth and a clear increase in pH in used culture medium compared to the control culture (Figure 3). The

strongest effect was seen after SA addition, from 9.7 g to 8.1 g cell mass and from pH 5 to pH 6.2.

In Treatment 2, the cell mass of Populus culture (Figure 4) was affected only by MeJA, which caused a decrease from 12.8 g to 10.6 g. In this culture, medium pH in the SA treated culture (pH 6.2) was lower than in the control (pH 6.5), but it was higher in the chitosan and MeJA treated cultures, pH 6.9 and pH 6.7, respectively.

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Figure 3 Cell mass and pH value of PS-S cultures after addition of different substances in Treatment 1. Averages from triplicate samples with standard deviations are shown.

Figure 4 Cell mass and pH values of Populus cell cultures after addition of different substances in Treatment 2. Averages from triplicate samples with standard deviations are shown.

Figure 5 Cell mass and pH value of Populus cell cultures after additions of different substances in Treatment 3. Values are from only one sample for each treatment.

4,5 5,0 5,5 6,0 6,5 7,0 0 2 4 6 8 10 12

1-PS-untr 1-PS-NIC 1-PS-NIA 1-PS-SA

A ve rag e p H o f c u ltu re m e d iu m A ve ra ge cel l m ass (g ) Cell cultures Mass pH 6,0 6,2 6,4 6,6 6,8 7,0 7,2 0 2 4 6 8 10 12 14

2-PO-untr 2-PO-SA 2-PO-chi 2-PO-MeJA

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3.2 Production of phenolic compounds

In PS-S culture medium from Treatment 1 the content of phenolic substances seemed to increase by all treatments, although not to a significant degree (Figure 6).

In cell extracts from the same cultures (Figure 6), no differences were seen, except for a small decrease caused by SA.

When looking at total content of phenolic substances in the cultures (Figure 7), a clear lowering effect can be seen after SA addition, dependent partly on the lower cell mass of this culture. Figure 7 also illustrates that the major part (64 - 82 %) of the phenolic substances were found in the cell material.

Figure 6 Contents of phenolic compounds in PS-S cultures after addition of different

substances in Treatment 1. Averages from triplicate samples with standard deviations are shown.

Figure 7 Production and distribution of phenolic compounds in PS-S cultures in Treatment 1. 0 5 10 15 20 25 30 35 40 45 50 55 60

Ph en o lic c o n te n ts (µ g/ ml ) PS-S culture medium 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0

Phe no lic con te nts ( µ g/m g) PS-S cells 0 2 4 6 8 10 12

Phe no lic pr o du ct io n (m g) PS-S cultures

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In Treatment 2, no effects on phenolic content in the culture medium from the Populus culture were observed (Figure 8). However, in the cell material a large increase in phenolics was measured in extracts from MeJA treated cultures, from 0.7 µg/mg to 6 µg/mg, which represents a 8.5 times increase.

The distribution of phenolic compounds between culture medium and cell material is illustrated in Figure 9. Approximately 90 % of the phenolics were found in the cell extracts from the control, SA-treated and chitosan-treated cultures, while in the MeJA-treated culture the large increased amounts of phenolic substances produced were retained in the cells, i.e. 98 % of total phenolics found in the cell material.

Figure 8 The total phenolic contents in Populus cell cultures after addition of different

substances in Treatment 2. Averages from triplicate samples with standard deviations are shown.

Figure 9 Production and distribution of phenolic compounds in Populus cultures in Treatment 2. Averages from triplicate samples with standard deviations are shown.

In Treatment 3, the content of phenolic substances in the medium from the Populus culture (Figure 10) was increased by some of the treatments. SA caused the largest increase from 17 µg/ml to 52 µg/ml. This increase seemed to be partially inhibited by a previous addition of

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Ph e n o lic c o n te n ts (µ g/ m l)

Populus culture mediums

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 P h en oli c co n te n ts (µg/m g) Populus cells 0 10 20 30 40 50 60 70 80

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NIC, resulting in a content of 35 µg/ml. On the other hand, a combination of NIC and

chitosan resulted in higher phenolic content than either NIC or chitosan alone. By themselves they did not differ much from the control. Also a combination of NIC and MeJA gave a higher content than the control.

Similarly there was an increased phenolic content also in cell extracts (Figure 10) after SA addition. The most prominent effects on phenolics in cell extracts were caused by NIC and MeJA in combination, 2.8 µg/mg compared to the control value 0.9 µg/mg.

The distribution of phenolic substances between culture medium and cells (Figure 11) followed approximately the same pattern as for cell extracts as such. In this experiment, like in the other two, the highest quantity of phenolic substances was found in the cell material.

Figure 10 Phenolic contents in Populus culture after addition of different substances in Treatment 3. Values from only one sample for each elicitation are shown.

Figure 11 Production and distribution of phenolic compounds in Populus cultures, Treatment 3. Values from only one sample for each elicitation are shown.

0 10 20 30 40 50 60 Ph e n o lic c o n te n t ( µ g/ m l)

Populus culture mediums

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3.3 Effects on Bacterial growth

Plant cell extracts were tested for effects on bacterial growth. One reason to choose extracts rather than culture medium was that most of the phenolic production in cell cultures was found in the cells. Another reason was that plant extract is the major study objective in most of other relevant researches so that the outcome of this project could be compared with results of other studies.

3.3.1 Agar plate assay

Cell extracts from Treatment 1

Among the four PS-S cell extracts from Treatment 1, the 1-PS-untr and the 1-PS-SA were first selected for tests on bacteria since the addition of SA resulted in the largest differences in cell mass, pH, and phenolic production compared to the untreated.

The two extracts had a concentration of 0.1 mg/µl and were tested on B. subtilis cultured on agar plates. After overnight incubation no inhibitory zone was observed.

The four PS-S cell extracts were concentrated to 0.25 mg/µl. All four extracts were tested on

B. subtilis cultured on agar plates. After overnight incubation no inhibitory zone was observed.

Among the four Populus cell extracts from Treatment 2, the 2-PS-untr and the 2-PS-MeJA were tested first on B. subtilis agar plates because the addition of MeJA resulted in an extra high production of phenolic compounds compared to that of the untreated one.

The two extracts had a concentration of 0.1 mg/µl. After overnight incubation no inhibitory zone was observed.

The four Populus cell extracts were concentrated to 0.25 mg/µl and tested on B. subtilis agar plates. After overnight incubation inhibitory zones were observed. The size of the inhibitory zones are shown in Figure 12.

The samples with the highest dose, 7.5 mg, all showed inhibitory effects on bacteria B.

subtilis. The 2-PO-MeJA and the 2-PO-SA cell extracts both resulted in a 10.5 mm of

inhibitory zone, and 2-PO-untr gave 7.5 mm. With medium dose, 5 mg, the 2-PO-chi and the 2-PO-SA cell extracts had inhibitory zones of 7.5 mm and 8.5 mm respectively. The lowest dose, 2.5 mg, and the negative control gave no inhibitory effects.

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Figure 12 Size of inhibitory zones by Populus cell extracts on B. subtilis cultured agar plates. The cell extracts were from Treatment 2.The dose of 2.5 mg and the negative control gave no inhibitory effects.

Figure 13 Photo of a B. subtilis cultured agar plate. Two inhibitory zones were observed around two wells marked as 5(mg) and 7.5(mg). The sample was a cell extract of the 2-PO-SA.

6,5 7 7,5 8 8,5 9 9,5 10 10,5 11

D iam e te rs o f i n h ib ito ry z o n e s (m m ) (Di am e te r o f we lls was 6.5 m m )

Populus cell extracts

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The seven Populus cell extracts from Treatment 3 had a concentration of 0.4 mg/µl and were tested on B. subtilis cultured agar plates. After overnight incubation inhibitory zones were found on five of the seven agar plates. The sizes of the inhibitory zones are shown in Diagram A of Figure 14. Negative controls gave no inhibitory effect.

Cell extracts from the following cultures showed antibacterial effects: the PO-untr, the 3-PO-NIC, the 3-PO-SA, the 3-PO-chi, and the 3-PO-NIC+chi, with inhibitory zones between 7.5 and 10 mm. The 3-PO-SA cell extract resulted in inhibitory effects with all doses. The lowest dose of the other four samples did not show any inhibitory effect.

This test was repeated nine days later. Inhibitory zones were observed again, and still from the five samples. The sizes are shown in Diagram B of Figure 14.

In the repeated test the 3-PO-SA and the 3-PO-NIC+chi samples showed inhibitory effects at medium and higher doses, with inhibitory zones between 7 and 10 mm. None of the lower doses showed any inhibitory effect this time.

Figure 15 shows photo of two B. subtilis cultured agar plates. The inhibitory zones were caused by the 3-PO-SA cell extract. The left-hand plate was from the first test and the right-hand one was from the repeated test. The inhibitory zones on the right-right-hand plate are not as clear as the ones in the left-hand due to lack of back light.

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Figure 14 Size of inhibitory zones by Populus cell extracts on B. subtilis cultured agar plates. The cell extracts were from Treatment 3. Diagram A: the first test. Diagram B: the repeated test.

Figure 15 Photos of two B.subtilis cultured agar plates. Inhibitory zones were observed around three wells marked as 10(µl), 20(µl) and 30(µl) on the left-hand plate, and around two wells marked as 20(µl) and 30(µL) on the right-hand. The left-hand plate was from the first test and the right-hand from the repeated test. The samples were the 3-PO-SA cell extracts.

6,5 7 7,5 8 8,5 9 9,5 10 10,5 11 11,5 D iam e te rs o f i n h ib ito ry z o n e s (m m ) (Di am e te r o f we lls was 6.5 m m )

Negative control 4 mg 8 mg 12 mg A 6,5 7 7,5 8 8,5 9 9,5 10 10,5 11 11,5 D iam e te rs o f i n h ib ito ry z o n e s (m m ) (Di am e te r o f we lls was 6.5 m m )

(Repeated 9 days later)

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3.3.2 Microtiter 96-well plate assay

Based on the outcomes of agar plate assay the seven Populus cell extracts from Treatment 3 were concentrated to 5 mg/µl and tested on B. subtilis with the microtiter 96-well plate assay. Figure 16 shows a photo of one microtiter plate test.

The growth curves of bacteria B. subtilis presented in Figures 17- 22 are based on average values. Each figure includes growth curves of the bacteria exposed to an cell extract from a treated culture v.s the 3-PO-untr cell extract, both in two different doses, and the growth curve of the bacteria in the control culture.

In Figure 17, the curve with 50 mg dose of 3-PO-chi cell extract run below the curve of the culture control from the 5th hour to the end of the incubation period. The other three curves stayed above that of the culture control all the time. Moreover the two curves from exposure to 3-PO-untr were above the other three from the 4th hour.

Figure 18 shows a similar situation for bacteria growth exposed to the 3-PO-NIC+chi. The curve with 50 mg dose of 3-PO-NIC+chi cell extract went below the one of the culture control from the 5th hour to the end. The two curves from exposure to the 3-PO-untr were above the other three from the 4th hour.

This indicates that the increased dose of 3-PO-chi or 3-PO-NIC+chi cell extract inhibited the bacterial growth while the two samples in lower dose and 3-PO-untr samples in both doses stimulated the bacteria growth by different degrees.

The findings shown in Figures 17 and 18 are consistent with the observed results from the microtiter plate, Columns 7 and 9, in Figure 16.

In Figure 19 the two growth curves of bacteria exposed to the 3-PO-NIC+MeJA cell extracts went below the curves of bacteria exposed to the 3-PO-untr cell extracts from the 5th hour to the end but slightly over the curve of the culture control. The two curves of the

NIC+MeJA started from a higher OD than the other three curves which shows that the 3-PO-NIC+MeJA cell extract may contain some kinds of compounds causing a higher absorption at the wave length. The 3-PO-NIC+MeJA cell extract did not inhibit the bacterial growth, nor did it stimulate it.

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Figure 17 Growth curves of B. subtilis exposed to the 3-PO-chi and the 3-PO-untr cell extracts. The culture control is included.

Figure 18 Growth curves of B. subtilis exposed to the 3-PO-NIC+chi and the 3-PO-untr cell extracts. The culture control is included.

Figure 19 Growth curves of B. subtilis exposed to the 3-PO-NIC+MeJA and the 3-PO-untr cell extracts. The culture control is included.

0,2 0,4 0,6 0,0 2,0 4,0 6,0 8,0 10,0 12,0 OD Time (h)

Culture control 3-PO-untr, 25mg 3-PO-untr, 50mg 3-PO-chi, 25mg 3-PO-chi, 50mg 0,2 0,4 0,6 0,0 2,0 4,0 6,0 8,0 10,0 12,0 OD Time (h)

Culture control 3-PO-untr, 25mg 3-PO-untr, 50mg 3-PO-NIC+chi, 25mg 3-PO-NIC+chi, 50mg 0,2 0,4 0,6 0,0 2,0 4,0 6,0 8,0 10,0 12,0 OD Time (h)

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Figure 20 Growth curves of B. subtilis exposed to the 3-PO-NIC and the 3-PO-untr cell extracts. The culture control is included.

Figure 21 Growth curves of B. subtilis exposed to 3-PO-SA and 3-PO-untr cell extracts. The culture control is included.

Figure 22 Growth curves of B. subtilis exposed to the 3-PO-NIC+SA and the 3-PO-untr cell extracts. The culture control is included.

0,2 0,4 0,6 0,8 1,0 1,2 0,0 2,0 4,0 6,0 8,0 10,0 12,0 OD Time (h)

Culture control 3-PO-untr, 25mg 3-PO-untr, 50mg 3-PO-NIC, 25mg 3-PO-NIC, 50mg 0,2 0,4 0,6 0,8 1,0 0,0 2,0 4,0 6,0 8,0 10,0 12,0 OD Time (h)

Cultrue control 3-PO-untr, 25mg 3-PO-untr, 50mg 3-PO-SA, 25mg 3-PO-SA, 50mg 0,2 0,4 0,6 0,8 1,0 0,0 2,0 4,0 6,0 8,0 10,0 12,0 OD Time (h)

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4. Discussion

The outcome of this study is discussed from different angles in this chapter.

4.1 Cell growth, pH and stress response

Changes in cell mass and pH in the culture medium can give information regarding stress response in plant cell cultures. In comparison with the 1-untr culture the cell mass of PS-S decreased slightly in all three cultures after the stress signalling compounds were added (Figure 3). In most cases the cell mass from treated Populus cultures decreased to a different extent (Figure 4-5). This indicates that the plant cell culture responded to the stress caused by the added substances. The response made the culture reallocate its basic capacity for growth or other cell functions as an effect of growth-defense tradeoffs (Huot et al. 2014). A part of such capacity could then be used to activate its defense mechanism. For example, growth was inhibited while the production of phenolic substances was increased in the MeJA treated cell cultures 2-PO-MeJA and 3-PO-NIC+MeJA.

Change of pH in the cultures varied to a large extent. Culture medium pH was clearly influenced by SA, increasing in PS-S culture and decreasing in Populus cultures. The opposite reactions may be due to the large difference in tissue differentiation between the cultures. However, the effects indicate distinct responses to SA addition. McCue et al. (2000) found that SA in combination with a low environmental pH stimulated phenolic production in peas. In the present study, the 1-PS-SA culture had a decreased production of phenolic

compounds with an increased pH in comparison to the 1-PS-untr culture. The 3-PO-SA culture had an increased phenolic production with a decreased pH in comparison to the 3-PO-untr culture. If McCue et al. are correct, it would be possible that the higher pH caused the SA effect of lower phenolic production in PS-S. Or, the lowered pH might have contributed to the SA effect of stimulating phenolic biosynthesis in Populus.

4.2 Phenolic production and defense response

A broad spectrum of defense related substances in plants can be found within the phenolic metabolism. The analysis of total phenolics is therefore often used as a measure of plant stress response. The most prominent effect on phenolic content in the cultures was seen after MeJA treatment (Figures 8 and 9). Also SA addition to the Populus culture resulted in increased phenolic content (Figure 10). The increased phenolic content caused by MeJA and SA is in line with the inhibitory effects on bacterial growth in the agar plate assay (Figures 12, 14) by extracts from the treated cultures.

4.3 Added compounds and defense response

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The elicitor chitosan did not show any clear effect on phenolic production or defense response for Populus. In accordance with this, the culture grew well. However, extracts from chitosan treated cultures showed a clear inhibiting effect on bacterial growth (Figures 12, 17) which illustrates that the analysis of total phenolics is not always a reliable method to indicate defense induction in plant cells. Other pathways within secondary metabolism might have been induced by chitosan.

4.4 The NIC effect

The effect of NIC in this study was interesting, because it appeared to give rise to substances that stimulated bacterial growth in the microtiter plate assay (Figure 20). In combination with SA, NIC also caused increased growth, while SA itself did not influence growth considerably in this assay (3-PO-NIC+SA; Figure 22). In the agar plate assay, NIC erased the inhibitory effect of SA. The NIC+SA culture also had lower phenolic production than the 3-PO-SA culture. These results show that NIC weakened the response caused by 3-PO-SA.

The situation was similar for the 3-PO-NIC+chi culture. NIC counteracted the chitosan effect on culture pH, growth and phenolic production. However, in the microtiter plate assay,

extracts from the NIC treated culture could not override the growth inhibiting effect of extract from chitosan treated culture.

The purpose of using NIC was to prepare, or "prime" the cells so they would respond more strongly to the following treatments. Obviously the cultures responded, even though the results from the parameters analyzed here instead pointed towards an opposite response, which is a new finding regarding effects of NIC.

4.5 Cell extracts and bacterial growth

We should keep in mind that the two growth assays used had different outcomes. In the agar plate assay a solid growth medium is used and an endpoint result is recorded. In the microtiter plate assay a liquid medium is used and the growth can be followed continuously. Cell

extracts from untreated as well as treated Populus cultures all showed inhibitory effects on Gram-positive bacteria in the agar plate assay. In the microtiter plate assay, the clearest results were growth inhibition by extracts from chitosan cultures and growth stimulation by extracts from NIC cultures, when compared to extracts from untreated cultures. MeJA was not tested alone, but in combination with NIC it resulted in growth inhibition.

According to a study by Nostro et al. (2012), plants from nature may have even stronger inhibitory effects than the cultured plant cells. If this is true also for Populus, the natural plant of Populus should in the future studies be tested on positive bacteria and also on Gram-negative bacteria.

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4.6 Defense compounds and antibacterial effects

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5. Conclusions

This is an initial study regarding production of antimicrobial substances by plant cell cultures. Treatment of Populus trichocarpa fine suspension cell cultures with elicitors and stress signal substances was performed to increase the production.

The following main conclusions can be drawn:

Cell culture growth and culture media pH as well as the production of phenolic substances were influenced by the treatments, which indicate stress responses of the P. trichocarpa

culture. This kind of responses can often be connected to the production of defense substances, for example antimicrobial compounds.

Extracts from untreated P. trichocarpa cultures could inhibit growth of B. subtilis on agar plates.

The production of bacterial growth inhibiting compounds was influenced by treatment of

P.trichocarpa cell cultures with elicitors and stress signal substances:

- inhibition of B. subtilis growth on agar plates by salicylic acid, methyl jasmonate and chitosan treatments.

- inhibition of B. subtilis growth in liquid media by chitosan treatment. - stimulation of B. subtilis growth in liquid media by nicotinamide treatment.

These results point at a possible strategy for production in plants or plant cell cultures of natural and non-toxic substances with antimicrobial properties.

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6. Future studies

Bacterial growth curve

In the microtiter plate assay the growth curve did not show that the bacteria in the control culture had grown much. However, a certain bacterial growth was clearly visible. There was also an aggregation of bacteria at the bottom of the culture control wells. It is possible that the measured values were too low due to some technical problems of the plate reader. This

experiment should be repeated with an accurate plate reader.

Qualitative results

The outcome of the microtiter plate assay did not provide the expected quantitative result. All results in this study are qualitative. A continuation of this study should establish the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Populus cell extracts on Gram-positive bacteria in agar medium.

MeJA v.s NIC+MeJA

MeJA was not used as a single addition in Treatment 3. Therefore cell extract from MeJA treated Populus culture could not compare with NIC+MeJA effects in agar plate assay and microtiter plate assay. This should be done in future studies.

Bacteria exposed to extracellular substances

The plant cell culture medium was not tested on bacteria in this project. The culture medium is, however, an interesting object for future studies as it could be a convenient source of excreted products in case of scale-up of the plant cell culture system.

Morphological investigation

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Appendixes

Appendix 1 The modified MS medium

Medium stocks: Part 1: 1 L

Chemicals Amount Unit

CaCl2·2H2O 4.44 g KH2PO4 3.4 g Na2EDTA·2H2O 0.37 g MnSO4·H2O 0.17 g ZnSO4·7H2O 0.09 g H3BO3 0.06 g KI 8 mg Na2MoO4·2H2O 2.5 mg Deionized water Up to 1 L Part 2: 1 L

Chemicals Amount Unit

KNO3 45.9 g NH4NO3 9.09 g MgSO4·7H2O 3.7 g FeSO4·7H2O 0.28 g Deionized water Up to 1 L Part 3: 1 L

Compounds Amount Unit

m-inositol 10 g Thiamine 0.05 g Pyridoxine 0.05 g Nicotinamide (niacin-amide) 0.1 g Deionized water Up to 1 L Part 4: 2 L

Amino acides (L-forms) Amount Unit

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41 Lysine 0.39 g Methionine 0.01 g Phenylalanine 0.01 g Proline 0.39 g Serine 2.55 g Threonine 0.82 g Tyrosine 0.01 g Valine 0.46 g Deionized water Up to 2 L Hormones stocks

Hormones Stock concentration Solvent

2,4-D 1 mg/ml Ethanol

Kinetin 1 mg/ml Deionized water

BA (benzyladenine) 0.4 mg/ml Deionized water

GA3 (gibberellic acid) 1 mg/ml Deionized water

To make 10 L of the modified MS medium: 1. Mix the stocks: part 1 1 L

part 2 1L part 3 100 ml part 4 100 ml 2. Add sucrose 300 g.

3. Fill up with deionized water to 10 L. 4. Adjust pH to 6.0 with 1M NaOH.

5. Add 2,4-D stock 10 ml and kinetin stock 200 µl for medium I, or BA stock 5 ml and GA3 stock 3.5 ml for medium III GA3.

6. Check and adjust pH.

7. (Add 80 g of agar if agar medium is needed.)

8. Distribute the medium to smaller Erlenmeyer flasks with 50 ml or 100 ml per flask. 9. Autoclave the medium for 20 minutes at 121 oC.